Method and apparatus for encoding cellular spatial position information

ABSTRACT

A system, methods, and apparatus are described to collect and prepare single cells and groups of cells from microsamples of specimens and encode spatial information of the physical position of the cells in the specimen. In some embodiment, beads or surfaces with oligonucleotides containing spatial barcodes are used to analyze DNA or RNA. The spatial barcodes allow the position of the cell to be defined and the nucleic acid sequencing information, such as target sequencing, whole genome, gene expression, used to analyze the cells in a microsample for cell type, expression pattern, DNA sequence, and other information, in the context of the cell&#39;s physical position in the specimen. In other embodiment, markers such as isotopes are added to a microsample to encode spatial position with mass spectoscopy or other analysis. The spatial encoded information is then readout by analysis such as DNA sequencing, mass spectrometry, fluorescence, or other methods.

CROSS REFERENCE TO RELATED APPLICATIONS (IF ANY)

This application claims the benefit of the priority date of provisionalpatent application 62/247,368, filed Oct. 28, 2015 (Jovanovich andWagner, “Method and apparatus for encoding cellular spatial positioninformation”), the contents of which are incorporated herein in theirentirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT (IF ANY)

None.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT IF THE CLAIMEDINVENTION WAS MADE AS A RESULT OF ACTIVITIES WITHIN THE SCOPE OF A JOINTRESEARCH AGREEMENT

None.

REFERENCE TO A “SEQUENCE LISTING”

None.

BACKGROUND OF THE INVENTION

Analysis of single cells and groups of cells is now beginning to provideinformation to dissect and understand how cells function individuallyand unprecedented insight into the range of individual responsesaggregated in ensemble measurements. Single cell methods forelectrophysiology, flow cytometry, imaging, mass spectrometry (Lanni, E.J., et. al. J Am Soc Mass Spectrom. 2014; 25(11):1897-907.), microarray(Wang L and K A Janes. Nat Protoc. 2013; 8(2):282-301.), and NextGeneration Sequencing (NGS) (Saliba A. E., et. al. Nucleic Acids Res.2014; 42(14):8845-60.) have been developed and are driving an increasedunderstanding of fundamental cellular processes, functions, andinterconnected networks. As the individual processes and functions areunderstood and differentiated from ensemble measurements, the individualinformation can in turn lead to discovery of how network processes amongcells operate. The networks may be in tissues, organs, multicellularorganisms, symbionts, biofilms, surfaces, environments, or anywherecells interact.

Next Generation Sequencing (NGS) of single cells is rapidly changing thestate of knowledge of cells and tissue, discovering new cell types, andincreasing understanding of the diversity of how cells and tissuefunction. Single cell NGS RNA sequencing (Saliba A. E., et. al., NucleicAcids Res. 2014; 42(14):8845-60.) (Shapiro E. et. al., Nat Rev Genet.2013; 14(9):618-30.) is unveiling the complexity of cellular expression,and the heterogenity from cell to cell, and from cell type to cell type(Buettner F. et. al., Nat Biotechnol. 2015; 33(2):155-60.). In situsequencing (Ke R et. al., Nat Methods. 2013; 10(9):857-60.), (Lee J H,et. al., Nat Protoc. 2015; 10(3):442-58.) (Lee J H, et. al., Science.2014, 21; 343(6177):1360-3.) has shown the feasability of directlysequencing of fixed cells. However, for RNA, many fewer reads aregenerated with in situ sequencing, biasing against detection of lowabundant transcripts. Photoactivatable tags have been used to capturemRNA from single cells (Lovatt D et. al., Nat Methods. 2014;11(2):190-6.) from known location in tissue, albeit with low throughputcapture and manual cell collection.

Single cell nucleic acid sequencing technology and methods using NGS andNext Next Generation Sequencing (NNGS) are rapidly evolving. Commoncomponents are incorporation of a marker or barcode for each cell andmolecule, reverse transcriptase for RNA sequencing, amplification, andpooling of sample for NGS and NNGS (collectively termed (N)NGS) librarypreparation and analysis. Starting with isolated single cells in wells,barcodes for individual cells and molecules have been incorporated byreverse transcriptase template switching before pooling and PCRamplification (Islam S. et. al. Genome Res. 2011; 21(7):1160-7.)(Ramsköld D. et. al. Nat Biotechnol. 2012; 30(8):777-82.) or on abarcoded poly-T primer with linear amplification (Hashimshony T. et. al.Cell Rep. 2012 Sep. 27; 2(3):666-73.) and unique molecular identifiers(Jaitin D. A. et. al. Science. 2014; 343(6172):776-9.).

Recent pioneering work has used the power of nanodroplets to performhighly parallel processing of mRNA from single cells with reversetransciption incorporating cell and molecular barcodes from freedprimers (inDrop) (Klein A. M. et. al. Cell. 2015; 161(5):1187-201.) orprimers attached to paramagnetic beads (DropSeq) (Macosko E. Z. et. al.Cell. 2015; 161(5):1202-14.). Both inDrop and DropSeq represent scalableapproaches that will change the scale from 100s of cells previouslyanalyzed to 1,000s and more. However, neither method encodes where inthe physical three dimensional (3D) tissue the cell originated and whatcells were in that microenvironment.

In the future, the spatial position of each individual cell (or groupsof cells in a microenvironment) and what the state of individual cell(s)is, i.e., DNA sequence, DNA modifications, RNA splicing, RNAmodifications, RNA expression pattern, proteome, metabolome, etc., willfurther the understanding of how tissue functions, how the diversity ofcells and the heterogeneity affects human health, including cancerformation and progression, how the microbiome interacts with the host,cell and tissue response to stimuli including therapeutic agents(Crosetto N. et. al. Nat Rev Genet. 2015; 16(1):57-66.), and how cellsinteract in the environment among other fundamental questions.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein is a Single Cell Spatial Analysis System™ 100 thatencodes the information of the spatial position 130 where a microsample125 was collected from tissue or from a specimen 301 for single cellsequencing, proteomics, cellular analysis, metabolomics and otheranalysis modalities. The Single Cell Spatial Analysis System 100 willgive researchers, clinicians, forensic scientists, and many otherapplications and disciplines, a fundamental new capability to understandhow single cells function, combining two and three dimensional spatialinformation with gene expression, and/or sequencing information,cellular response to chemical/enviromental challenges, proteinexpression, and other analysis methods.

The Spatial Subsystem (also referred to herein as the “SpatialPreparation Subsystem” or “Spatial Preparation System” or “SpatialPreparation Module”) 200, which collects microsamples 125, is anautomated approach to collecting and generating single cells or groupsof cells from microsamples 125 from matrices such as tissue withoutpainstaking, lower throughput, or labor intensive manipulations, e.g.,Laser Capture Microdissection (Datta S. et. al. Histol Histopathol. 2015Apr. 20:11622.), manual pipette collection (Morris J. et. al. 2011. J.Vis. Exp. (50), e2634,) (Kajiyama T. et. al. Plant Cell Physiol. 2015;56(7):1320-8.), or Fluidigm's low throughput C1 system(https://www.fluidigm.com/products/c1-system). Compared to in situsequencing, the Single Cell Spatial Analysis System allows the fullpower of current commercially available (N)NGS systems to be used forthe analysis engine.

The Single Cell Spatial Analysis System can encode the position of wherethe microsample 125 was located in the tissue or specimen 301 bydifferent methods for different analyses. For genomic analysis, theSingle Cell Spatial Analysis System 100 can use primer sets with nucleicacid barcodes for spatial position attached to beads or other surfacessuch as flow cells to encode the spatial position 130 into DNA. Forproteomic analysis by mass spectrometry, isotopes or other markers canbe added to the microsamples 125 to encode the position while for enzymeactivity assays fluorescent, Raman, optical, or other markers can beadded to encode the spatial position 130. For metabolomic analysis,isotopes, fluorescent, or other markers can be added.

This approach of encoding into the microsample 125 where it originatedin three dimensions from the specimen 301 is a fundamentally newapproach to prepare samples from single cells and to understand thegenetics, gene and protein regulation, metabolism of individual cells,how they function in a 3D tissue or biofilm structure, and what celltypes, including rare cells, are present and where among otherscientific, clinical, and applied information.

The Single Cell Spatial Analysis System 100 described can input raw,unprocessed samples, or other primary or secondary samples, and forgenomic analysis produce either cDNA or prepared DNA libraries ready forDNA sequencing. The Single Cell Spatial Analysis System 100 for genomicassays has additional advantages over existing technology. Automatedpreparation of samples for NGS typically starts with purified bulk DNAor RNA and only part of the workflow is integrated, such as librarypreparation with manual steps of QC and post-library preparationamplification if needed. Automated sample preparation has not yet beenintegrated from raw samples, such as blood, tissue samples, fine needleaspirates, and other samples, to libraries ready for sequencing andremains largely manual. No automated NGS library preparation systemshave yet been commercialized for single cells or for researchers andclinicians to routinely sequence cells from tissue. No system currentlyexists that collects single cells with positional information fornucleic acid analysis, or performs high throughput single cell RNA-Seq,or processes raw samples to libraries.

Disclosed herein is a system that can integrate one or more of theoverall steps to take samples from specimens (i.e., tissue, biofilms,and other multi-dimensional matrices with cells or viruses) and preparesingle cells, groups of cells, or cells and viruses (collectively orindividually referred to as cells) to produce samples with informationencoded about the cell's spatial positions in the original specimen. Thecell's spatial position in the original specimen can be encoded in amarker, e.g., a spatial barcode, which is added to the specimen, asubregion, a microsample, or other part of a specimen, in a manner thatencodes the single cell's position into the cell or components of thecell. Alternatively, the spatial position can be encoded by physicalposition of the sample as it is readout from a flow cell for NGS.

An instance of a spatial barcode for nucleic acids is described and oneembodiment illustrated in detail. Microsamples from a subregion of aspecimen are collected in physical order, such as a raster pattern or byrows or columns, and the microsamples placed in a known order into afluidic stream or fixed wells or onto a surface. The term “microsample”is used to mean the smallest portion of the specimen that is collectedas an individual sample that will be encoded with a single spatialmarker or barcode and is the smallest unit sampled from the specimen bythe system; microsamples may contain single cells to groups of cells.Single cells can be produced from the microsamples in microdrops, e.g.,nanodroplets or boluses generated with a paramagnetic bead which has anattached oligonucleotide with a unique spatial DNA barcode, a type ofspatial barcode, for the microsample. The beads with known uniquespatial DNA barcodes are added in known order to the microsamples, whichthereby will encode the order of the microsamples and cells in themicrosamples and produce spatially encoded single cells. Additionalbarcodes may be unique for each cell or molecule or have quality controlor other information. Nucleic acid is released from the single cellinside the nanodroplet or bolus and enzymology used to attach theoligonucleotide containing the spatial barcode to the cellular nucleicacid: this encodes the nucleic acid sequence to analyzed with thespatial DNA barcode on the oligonucleotide. After library preparationand DNA sequencing, the sequence of the spatial barcode is determinedalong with DNA or RNA sequence information. The spatial barcode, whichwas added in known order to microsamples that were also ordered in knownorder, can decode where in the specimen the microsample orginated. Twoand three dimensional spatial relationships of the microsamples andcells can then be determined and interpreted with the DNA or RNAsequence information to develop spatial information of what cells werepresent in the specimen, where the cells were located, the cell's DNAsequence and/or RNA expression, and other information.

Different embodiments of the Single Cell Spatial Analysis System canencode the spatial information for decoding by analytical methodscomprised of DNA sequencing, DNA microarrays, RNA sequencing, massspectrometry, Raman spectroscopy, electrophysiology, flow cytometry, andmany other analytical methods well known to one skilled in the artincluding multidimensional analysis. For nucleic acids, the spatialposition of where the cells originated from the specimen or tissue isencoded into the DNA sequence information, in one embodiment, byutilizing paramagnetic beads with a spatial barcode in an attachedoligonucleotide that is unique for each cell/bead combination. Thespatial barcode may be incorporated into the cDNA product by reversetranscriptase or RNA ligase if RNA is being analyzed, or into DNA by DNApolymerase or DNA ligase if DNA is being analyzed. The RNA from thespecimen is processed into cDNA and then into sequencing libraries for(N)NGS analysis, while preserving the information of the spatialorganization of the cells within the original specimen by attachment ofa spatial encoding nucleic acid barcode. In other instances, DNA isprocessed to a ready to sequence library; both RNA and DNA may beanalyzed from a single cell, or nucleic acid and other properties, suchas metabolites or proteomics, can be analyzed. The system described iscompatible with commercially available downstream library preparationand analysis by both NGS and NNGS sequencers. The term (N)NGS is used toconnote either NGS or NNGS sequencers or sample preparation methods asappropriate. For protein or metabolomic analysis, the Single CellSpatial Analysis System encodes the spatial position of the microsamplefor downstream analysis by adding barcodes comprising markers orisotopes. As contemplated herein, next generation sequencing (NGS) ornext-next generation sequencing (NNGS) refers to high-throughputsequencing, such as massivley parallel sequencing, (e.g., simultaneously(or in rapid succession) sequencing any of at least 100,000, 1 million,10 million, 100 million, or 1 billion polynucleotide molecules).Sequencing methods may include, but are not limited to: high-throughputsequencing, pyrosequencing, sequencing-by-synthesis, single-moleculesequencing, nanopore sequencing, semiconductor sequencing,sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumine),Digital Gene Expression (Helicos), Next generation sequencing, SingleMolecule Sequencing by Synthesis (SMSS) (Helicos), massively-parallelsequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing,Maxam-Gilbert or Sanger sequencing, primer walking, sequencing usingPacBio, SOLiD, Ion Torrent, Genius (GenapSys) or Nanopore (e.g., OxfordNanopore) platforms and any other sequencing methods known in the art.

The Single Cell Spatial Analysis System described can have multiplesubsystems and modules that perform processing or analysis. In apreferred embodiment, there is a subsystem, the Spatial PreparationSubsystem™, that inputs tissue and/or other specimens and outputsmicrosamples of single cell(s) encoded with cellular locationinformation which may be DNA barcodes or other encoding methods. In oneembodiment, the first module, the Spatial Sampler™ module, collectssamples from defined areas of specimen(s) to provide single cellsthrough a fluidic system in the order of their original spatialorientation to the second module, the Spatial Encoder™ module.

In one embodiment, cell imaging solutions, such as cell specificantibodies, stains, or other reagents, can be added to the tissue and anoptical or other imaging device scans the tissue in the Spatial Samplermodule. The optical cellular image information can be used to decidewhich part or subregions of the specimen should be analyzed or to gatherinformation to correlate with the downstream analysis. In otherembodiments, solutions comprised of antagonists, chemicals, biologicals,therapeutic drugs, or other compounds can be added to the tissue asneeded before sampling and analysis. The Spatial Sampler module can thenapply solution(s) to disassociate tissue or other specimens; as needed,the progression of dissociation can be monitored optically or by othermeans in the Spatial Sampler module. In one embodiment, for protein,metabolic, or other analysis, the Spatial Sampler module can applysolutions that encode the spatial position, comprised of combinations ofisotopes or fluors, or marker molecules not present in the specimen,that are later decoded to identify where in the specimen the microsampleoriginated.

In a preferred embodiment of the Spatial Sampler module, after tissuedissociation, a transfer device, such as a multifunctional head, canthen collect a layer from the specimen onto a surface such as a cellimpermeable transfer membrane with vacuum applied. The transfer devicecan be moved to an input device for a fluidic system which has one ormore fluidic channels, wells, or surfaces where microsamples from asubregion of the surface are transferred in physical positional orderinto the fluidic channel(s), well, or surface. When fluidic channels ormicrochannels are used, the microsamples are entrained into a fluidicflow in known order that can be tracked to the physical position of themicrosample in the original specimen. The channels may be capillaries,microchannels, micropipettes, or of other forms. Microsamples may betransferred one at a time or multiple samples transferred in parallel.In another embodiment, the input device directly samples the specimen.

In the Spatial Encoder module, for nucleic acids, the spatial positionof where the cells originated from the tissue is encoded into the DNAsequence information in one embodiment by utilizing paramagnetic beadswith an attached oligonucleotide with a spatial barcode unique for eachcell/bead combination. The spatial barcode can be incorporated into thecDNA product by reverse transcriptase if RNA is being analyzed or intoDNA from a primer by DNA polymerase or DNA ligase if DNA is beinganalyzed. In some instances the primer is attached to a solid surfacesuch as a paramagnetic bead, microparticles, fibers, channel, wall,flowcell, membranes, test tubes, pipette tips or microwells or othersurface. The term bead is used to encompass any solid surface, poroussurface, or other implementations without limitation including flatsurfaces.

In a preferred embodiment for nucleic acids, the Spatial Encoder moduleintegrates delivery of spatially barcoded beads entrained in known orderwith microdrops, e.g., nanofluidic droplet or bolus generation andsample preparation of DNA, RNA, or cDNA. The spatially encoded beadshave DNA or other barcodes that identify which microsample is beinganalyzed and, by knowing the order the microsamples were taken andsubsequent handling, the physical position of the microsample in thespecimen is encoded. In some embodiments, the spatially barcoded beadsare added using a microfluidic nozzle to generate microdrops(nanodroplets or in others boluses are generated from the microsample inan immiscible fluid with preferably one spatically barcoded bead perdroplet or bolus and only one cell. In some instances the nanodropletsor boluses may perform cellular lysis, mRNA binding, and cDNA reactionworkflows. In others, cellular lysis, DNA binding, and DNA amplificationreactions may be performed.

In some instances, a third module, the Spatial Librarian Subsystem (alsoreferred to as a “Spatial Librarian System” or “Spatial LibrarianModule”) prepares an (N)NGS library for nucleic acid analysis from themicrodrops (nanodroplets or boluses). In the Spatial LibrarianSubsystem, the nanodroplets or boluses are processed separately or inthe preferred embodiment the nanodroplets or boluses are pooled afterspatial encoding to process many single cells and microsamplessimultaneously. The Spatial Librarian module performs the necessarybiochemistry to add adapters as needed, prepare an (N)NGS sequencinglibrary, amplify as necessary, and perform quality control of thelibrary. In other embodiments, the analytical function, such as DNAsequencing, is incorporated to create a sample-to-answer system.

In one aspect provided herein is a system comprising: (i) a biologicalspecimen; and (ii) added to each of a plurality of differentmicrosamples from the biological specimen, a marker comprising spatialinformation that encodes the original spatial position of themicrosample within the biological specimen. In one embodiment thebiological specimen comprises human tissue, animal tissue, or planttissue, a biopsy, a cellular conglomerate, an organ fragment, anorganism, whole blood, bone marrow, biome, a biofilm, a fine needleaspirate or any other solid, semi-solid, gelatinous, or frozen threedimensional or two dimensional matrix of biological nature. In anotherembodiment the microsamples comprise a single cell or a plurality ofcells. In another embodiment the marker comprises a polynucleotide. Inanother embodiment the nucleic acid is bound to a membrane, chipsurface, bead, surface, flow cell, or particle or is indirectly boundvia an adapter molecule e.g., a complementary nucleic acid or a chemicalcrosslinker. In another embodiment the marker comprises a peptide,antibody, protein, small molecule, isotope such as lanthanide, Ramanmarker, mass tag, fluorescent or chemiluminescent probe. In anotherembodiment the microsamples are dissociated from the biologicalspecimen. In another embodiment the microsamples are entrained inmicrodrops in a fluidic stream. In another embodiment the microsamplesare supported by at least one substrate, e.g., a membrane.

In another aspect provided herein is a device for the analysis of abiological sample, the device comprising: a sample module configured toextract microsamples from a biological specimen; and a recipient moduleconfigured to receive the microsample biological specimen from thesample module for analysis. In one embodiment the recipient moduleperforms a downstream analysis selected from nucleic acid sequencing,next generation sequencing, next next generation sequencing, proteomic,genomic, gene expression, gene mapping, carbohydrate characterizationand profiling, lipid characterization and profiling, flow cytometry,imaging, microarray, metabolic profiling, functional, or massspectrometry or combinations thereof.

In another aspect provided herein is a device comprising: an elementselected from a membrane, filter, surface, capillary, microchannel,device, and microfabricated chip; and means to bring the element intodirect contact or close proximity to a biological specimen for thepurpose of labeling or extracting a plurality of microsamples in anorder based on their original spatial position within the biologicalspecimen.

In another aspect provided herein is a system comprising: a stage forsupporting a biological specimen; a device comprising an array ofmarkers comprised in beads, surfaces, flat or microfabricatedstructures; means for transferring the array of markers into or onto thebiological specimen at predetermined spatial positions.

In another aspect provided herein is a method comprising: adding, toeach of a plurality of different microsamples from a biologicalspecimen, a marker comprising spatial information that encodes theoriginal spatial position of the microsample within the biologicalspecimen. In one embodiment the method further comprises dissociatingthe microsamples from the biological specimen. In another embodiment themethod comprises adding the markers to the microsamples beforedissociating the microsamples from the biological specimen. In anotherembodiment the method comprises adding the markers to the microsamplesafter dissociating the microsamples from the biological specimen. Inanother embodiment each microsample comprises a single cell. In anotherembodiment each microsample comprises a plurality of cells. In anotherembodiment dissociating the microsamples comprises extracting themicrosamples in a raster pattern across the biological specimen. Inanother embodiment the microsamples are dissociated in a 3-D pattern. Inanother embodiment dissociating comprises contacting the biologicalspecimen with a membrane, applying vacuum to the membrane to hold alayer comprising the microsamples; and removing the microsamples held bythe membrane from the biological specimen. In another embodiment themethod comprises removing a second layer of the microsamples from thebiological specimen after a first layer is removed. In anotherembodiment the method further comprises moving the dissociatedmicrosamples into a fluidic stream. In another embodiment the methodcomprises the microsamples are moved into the fluidic stream in an ordercorrelated with their original spatial position in the biologicalspecimen. In another embodiment the method comprises microsamples areincorporated into microdrops (e.g., nanodroplets or boluses) in thefluidic stream. In another embodiment the method comprises themicrodrops contain one or more beads. In another embodiment the methodcomprises the beads are paramagnetic. In another embodiment the methodcomprises the beads are functionalized with oligonucleotides comprisingthe spatial information in the form of a nucleotide barcode. In anotherembodiment the method comprises the nucleotide barcode is unique foreach cell or group of cells in the microsample. In another embodimentthe method comprises the oligonucleotide comprises barcodes forcellular, molecular, or quality control purposes. In another embodimentthe method comprises the nucleic acid of the sample component includingbut not limited to groups of cells or single cells is enzymaticallycombined with the oligonucleotide of the bead. In another embodiment themethod comprises the nucleic acid is subjected to library preparationand nucleic acid sequencing. In another embodiment the method comprisesthe oligonucleotide further comprises a poly T tail, and the methodcomprises capturing mRNA molecules from the microsamples having a poly Ttail; and reverse transcribing the mRNA molecules to produce cDNAmolecules comprising the barcode where the barcode provides the spatialinformation. In another embodiment the method comprises theoligonucleotide further comprises a capture sequence complementary to atarget sequence, and the method comprises capturing DNA molecules fromthe microsample having the target sequence; and extending theoligonucleotide to produce a nucleic acid molecule having a copy of thetarget sequence and comprising the barcode, wherein the barcode providesthe spatial information. In another embodiment the method comprisesdissociating comprises contacting the biological sample with a celldissociation solution comprising at least one protease that digestsextracellular matrix. In another embodiment the method comprises the atleast one protease is selected from collagenases, elastase, trypsin,papain, hyaluronidase, chymotrypsin, neutral protease, clostripain,caseinase, neutral protease (Dispase®), DNAse, protease XIV. In anotherembodiment the method comprises the cell dissociation solution is in theform of a fluid, mist, fog, or aerosol applied to the biological sample.In another embodiment the method further comprises decoding the spatialinformation in the microsamples to determine the original spatialposition of each microsamples.

In another aspect provided herein is a method comprising: providing abiological specimen; collecting microsamples from each of a plurality ofdifferent spatial positions in the biological specimen; attaching tonucleic acids in each microsample a marker comprising a nucleic acidbarcode comprising spatial information that encodes the original spatialposition of the microsample within the biological specimen, therebyproducing spatial encoded nucleic acids; sequencing the spatial encodednucleic acids; and based on the spatial information attached to eachspatial encoded nucleic acids, determining the original spatial locationof the nucleic acid in the biological specimen. In one embodiment themethod comprises sequencing spatial encoding nucleic acids combined froma plurality of different microsamples in a single high throughputsequencing run.

In another aspect provided herein is a system having a graphical userinterface that presents, based on spatial information obtained frommicrosamples of a biological specimen, a graphical representation of thebiological specimen including original spatial position of a pluralityof polynucleotides or polypeptides in the biological specimen.

In another aspect provided herein is a spatial preparation systemconfigured to entrain in a fluidic stream a plurality of microsamplesfrom a biological specimen, wherein the microsamples are contained inspatially separated microdrops in a fluidic stream and positioned in anorder based on their original spatial position within the biologicalspecimen, wherein the system comprises: a) a spatial sampler subsystemconfigured to extract a plurality of microsamples from differentoriginal spatial positions in a biological specimen; and b) a spatialencoder subsystem comprising one or more spatial encoder microchannels,each having an inlet and an outlet; wherein the spatial samplersubsystem delivers the microsamples to the spatial encoder microchannelinlets in a predetermined order based on their original spatial positionin the biological specimen, and the spatial encoder subsystemincorporates the microsamples into spatially separated microdrops in afluidic stream. In one embodiment of the spatial preparation system: (i)the spatial sampler subsystem comprises: (1) a specimen holder, and (2)a multifunctional head comprising a transfer head comprising one or moreextraction channels, wherein the extraction channels communicate with aliquid source and, optionally, a gas source, each under positive and/ornegative pressure, and wherein the one or more extraction channelscomprise ends covered with one or more air permeable, cell impermeabletransfer membranes, and wherein, the multifunctional head is mounted ona three axis stage to position the multifunctional head to extract, bycontact adhesion or by vacuum, the microsamples from the specimen holderonto the one or more transfer membranes; and (ii) the spatial encodersubsystem comprises: (1) a microdroplet generator comprising a source ofimmiscible liquid in communication with each spatial encodermicrochannel at a junction, wherein mixture of the immiscible liquidwith the fluidic stream at the junction forms spatially separatedmicrodrops comprising the microsamples; and (2) optionally, amicrosample encoder assembly comprising a plurality of reservoirs, eachcomprising a different spatial marker and each communicating with thespatial encoder microchannel, and, optionally reservoirs comprising areactants sufficient to attach the tags to analytes in the microsamples,wherein different spatial markers are incorporated with microsamples indifferent microdrops. In another embodiment the multifunctional headfurther comprises a dispense head configured to dispense liquids, e.g.,imaging reagents or dissociation solution, onto the biological specimen.In another embodiment the transfer head comprises a plurality ofextraction channels where in the extraction channels are arrayed in atwo dimensional array (e.g., a line) or a three-dimensional array (e.g.,a plane). In another embodiment the spatial encoder subsystem comprisesa plurality of fluidic channels that merge into the encoder channel inwhich each has an inlet configured to receive the microsamples from anextraction channels. In another embodiment the transfer membranes haveattached thereto a plurality of capture elements, each capture elementcomprising a particle, which is optionally paramagnetic, having attachedthereto one or more antibodies that bind into cells in the biologicalspecimen, and nucleic acid markers comprising positional barcodescomprising spatial information where the spatial information calling tothe position of the particle on the multifunctional head. In anotherembodiment the nucleic acid markers further comprise cell markersidentifying the cell to which particle binds, and/or molecular barcodesthat differently label different nucleic acid molecules and a singlecell.

In another aspect provided herein is a spatial analysis systemcomprising: a) a spatial preparation subsystem as disclosed herein, andb) a spatial librarian subsystem configured to perform a series ofbiochemical reactions on an emulsion comprising microdrops produced bythe spatial preparation subsystem, wherein the spatial librariansubsystem comprises: a) a reaction device comprising an inlet configuredto receive microdrops from the spatial preparation subsystem, at leastone reaction chamber, and an outlet;b) a reagent rail communicating withthe reaction device through a microchannel and comprising reagentsufficient to perform at least one of biochemical reaction on analytesin the microdrops; and c) one or more pumps configured to move thereagents from the reagent rail through the microchannel to the reactionchamber of the reaction device. In another embodiment the spatiallibrarian subsystem further comprises: c) a temperature controllerconfigured to control temperature in the reaction chamber. In anotherembodiment the spatial librarian subsystem further comprising:c) amagnet configured to reversibly immobilize paramagnetic particlescontained in the reaction chamber. In another embodiment the biochemicalreactions comprise at least: (i) reverse transcription of messenger RNAinto cDNA; and (ii) amplification of cDNA. In another embodiment thebiochemical reactions comprise at least: (i) primer extension of aprimer hybridized to a DNA template to create an extension product; and(ii) amplification of the extension product.

In another aspect provided herein is a method comprising entraining in afluidic stream a plurality of microsamples from a biological specimen,wherein the microsamples are contained in spatially separated microdropsin the fluidic stream and positioned in an order based on their originalspatial position within the biological specimen. In one embodiment themethod comprises: a) providing a biological specimen; b) collectingmicrosamples from each of a plurality of different spatial positions inthe biological specimen; c) introducing the microsamples in apredetermined order into a fluidic stream in a fluidic channel; d)dividing the fluidic stream into microdrops by introducing boluses ofimmiscible liquid into the fluidic channel, whereby the microsamples areincorporated into microdrops that are spatially separated from eachother in the fluidic stream. In another embodiment the method furthercomprises: (i) introducing into the fluidic stream a plurality ofdifferent spatial markers encoding spatial information, wherein thedifferent spatial markers are incorporated into different microdrops inthe fluidic stream, thereby encoding each microdrop with spatialinformation. In another embodiment the analytes comprise nucleic acidsand the spatial markers comprise nucleic acids comprising nucleic acidbarcodes, wherein the method further comprises: (e) combining microdropsin a container in the form of an emulsion; (f) generating spatiallytagged nucleic acids by tagging nucleic acid analytes with the nucleicacid barcodes; (g) breaking the emulsion; (h) amplifying the taggednucleic acids. In another embodiment the analytes comprisepolyadenylated mRNA and the nucleic acid markers further comprise polyTtail, and generating spatially tagged nucleic acids comprises:hybridizing the polyT tail to polyadenylated mRNA nucleic acid markersto the mRNA molecules barcodes and reverse transcribing thepolyadenylated messenger RNA to produce that spatially tagged cDNAmolecules; performing second strand synthesis on the spatially taggedcDNA molecules to produce tagged double stranded cDNA molecules. Inanother embodiment the analytes comprise DNA molecules and the nucleicacid markers further comprise a nucleotide sequence complementary to atarget sequence, and generating spatially tagged nucleic acidscomprises:hybridizing the complementary nucleotide sequence to a targetsequence in the nucleic acid molecules and extending the nucleic acidmarkers to produce a double-stranded DNA molecule. In another embodimentthe method further comprises: applying imaging reagent to the biologicalsample; imaging the biological sample to which the imaging reagent hasbeen applied; based on the imaging selecting features of interest atpredetermined spatial positions in the biological sample; and extractingthe microsamples including the selected features of interest. In anotherembodiment the method further comprises, based on spatial informationencoded in the microsamples, determining the initial spatial position ofthe selected features in the biological specimen.

In another aspect provided herein is an apparatus, composition ofmatter, or article of manufacture, and any improvements, enhancements,and modifications thereto, as described in part or in full herein and asshown in any applicable Figures, including one or more features in oneor more embodiment.

In another aspect provided herein is an apparatus, composition ofmatter, or article of manufacture, and any improvements, enhancements,and modifications thereto, as described in part of in full herein and asshown in any applicable Figures, including each and every feature.

In another aspect provided herein is a method or process of operation orproduction, and any improvements, enhancements, and modificationsthereto, as described in part or in full herein and as shown in anyapplicable Figures, including one or more feature in one or moreembodiment.

In another aspect provided herein is a method or process of operation orproduction, and any improvements, enhancements, and modificationsthereto, as described in part or in full herein and as shown in anyapplicable Figures, including each and every feature.

In another aspect provided herein is a product, composition of matter,or article of manufacture, and any improvements, enhancements, andmodifications thereto, produced or resulting from any processesdescribed in full or in part herein and as shown in any applicableFigures.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 shows an overview of the overall workflow to generate single cellspatial information from specimens.

FIG. 2 shows an overview of a Single Cell Spatial Analysis System.Tissue or other specimens are converted to single cells and encoded withspatial information.

FIG. 3 shows an overview of a Single Cell Spatial Analysis Systemconfigured for nucleic acid sequencing. Specimens are converted tosingle cells and encoded with spatial information in the SpatialPreparation Subsystem for integrated NGS library preparation in theSpatial Librarian Subsystem.

FIG. 4 shows modules of the Spatial Preparation Subsystem configured fornucleic acid sequencing. The Spatial Sampler module collects cells fromdefined areas and dispenses them in known order into the Spatial Encodermodule that encodes the original position within the tissue into DNAusing unique primers as barcodes attached to beads. The SpatialPreparation Subsystem can output samples of single cells encoded withspatial DNA barcodes that are ready for library preparation.

FIG. 5 shows a Single Cell Spatial Analysis System configured forproduction of nucleic acid spatial libraries.

FIG. 6 shows a multifunctional head from a side view and a bottom viewwith an optical head, dispense head, and a changeable transfer membrane.The insert of the right of the figure shows the transfer membrane mayhave individually addressable subregions.

FIG. 7a shows an example of a multifunctional head with transfermembrane contacting a specimen. FIG. 7b shows an example of amultifunctional head with a transfer membrane about to transfer part ofa specimen to an input device with two arrays of microchannels.

FIG. 8a shows a bottom view of a multifunctional head with a transferredspecimen showing 12 microsamples transferred into input microchannelsand illustrating a subregion. FIG. 8b shows a bottom view of amultifunctional head with a transferred specimen showing 24 microsamplestransferred into input microchannels.

FIG. 9 shows an example of a single channel fluidic circuit of the inputdevice to input a subregion of the specimen into a fluidic flow.

FIG. 10 shows an example of a two channel fluidic circuit of the inputdevice that inputs two subregions of the specimen and combines them inknown order into a single fluidic flow.

FIG. 11a shows a capillary connector, valve, and router. FIG. 11b showsthree 75 um ID capillaries mounted in a FC connector. FIG. 11c shows asingle capillary in a FC connector covered with filter paper as atransfer membrane.

FIG. 12 shows 12 200 um OD capillaries arrayed in a linear connector.

FIG. 13a is an illustration of a bead with an oligonucleotide attachedthat contains a spatial DNA barcode. FIG. 13b is an illustration of asurface with an oligonucleotide attached that contains a spatial DNAbarcode.

FIG. 14 shows a single channel fluidic design of a Spatial Encodermodule that has four spatial barcodes.

FIG. 15 shows a single channel fluidic design of a Spatial Encodermodule with a spatial encoder reagent syringe pump that can addadditional reagents to a bolus or a microsample.

FIG. 16 illustrates a Spatial Librarian embodiment with a reagent railand reaction chamber with temperature control, optical interrogation,paramagnetic bead purification, and quality control analysisfunctionality.

FIG. 17 shows the direct transfer of spatial markers from an array ontothe specimen.

FIG. 18 shows an array of beads with known spatial barcodes andantibodies against cell surface markers attached to a surface.

FIG. 19 shows an example workflow encoding spatial information into DNAfrom polyadenylated mRNA.

FIG. 20 illustrates the capture and molecular biology to encode spatialinformation into DNA from polyadenylated mRNA.

FIG. 21 shows an example workflow of library preparation from spatiallyencoded double stranded DNA.

FIG. 22 illustrates a workflow to spatially encode genomic DNA fromsingle cells from a specimen.

FIG. 23 illustrates a workflow to spatially encode targeted regions ofgenomic DNA from single cells from a specimen.

FIG. 24 shows an example of using transposons to produce a sequencinglibrary from double stranded DNA with spatial encoding from single cellsfrom a specimen.

DETAILED DESCRIPTION OF THE INVENTION

NGS information, mass spectrometry and other modern high-throughputanalysis systems have revolutionized life and medical sciences. However,these and other high-throughput analysis systems fail to retain spatialinformation about where in the specimen the sample or microsampleoriginated. It is anticipated that single cell spatial information, orspatial information from groups of cells, of genomic, proteomicincluding protein expression, carbohydrate, lipid, and metabolism ofindividual cells will provide fundamental scientific knowledge andrevolutionize new research and clinical capacities.

All patents, patent applications, published applications, treatises andother publications referred to herein, both supra and infra, areincorporated by reference in their entirety. If a definition and/ordescription is set forth herein that is contrary to or otherwiseinconsistent with any definition set forth in the patents, patentapplications, published applications, and other publications that areherein incorporated by reference, the definition and/or description setforth herein prevails over the definition that is incorporated byreference.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Specimen: The term “specimen,” as used herein, refers to an in vitrocell, cell culture, virus, bacterial cell, fungal cell, plant cell,bodily sample, or tissue sample that contains genetic material. Incertain embodiments, the genetic material of the specimen comprises RNA.In other embodiments, the genetic material of the specimen is DNA, orboth RNA and DNA. In certain embodiments the genetic material ismodified. In certain embodiments, a tissue specimen includes a cellisolated from a subject. A subject includes any organism from which aspecimen can be isolated. Non-limiting examples of organisms includeprokaryotes, eukaryotes or archaebacteria, including bacteria, fungi,animals, plants, or protists. The animal, for example, can be a mammalor a non-mammal. The mammal can be, for example, a rabbit, dog, pig,cow, horse, human, or a rodent such as a mouse or rat. In particularaspects, the tissue specimen is a human tissue sample. The tissuespecimen can be, for example, a blood sample. The blood sample can bewhole blood or a blood product (e.g., red blood cells, white bloodcells, platelets, plasma, serum). The specimen, in other non-limitingembodiments, can be saliva, a cheek, throat, or nasal swab, a fineneedle aspirate, a tissue print, cerebral spinal fluid, mucus, lymph,feces, urine, skin, spinal fluid, peritoneal fluid, lymphatic fluid,aqueous or vitreous humor, synovial fluid, tears, semen, seminal fluid,vaginal fluids, pulmonary effusion, serosal fluid, organs,bronchio-alveolar lavage, tumors, frozen cells, or constituents orcomponents of in vitro cell cultures. In other aspects, the tissuespecimen is a solid tissue sample or a frozen tissue sample. In stillfurther aspects, the specimen comprises a virus, bacteria, or fungus.The specimen can be an ex vivo tissue or sample or a specimen obtainedby laser capture microdissection. The specimen can be a fixed specimen,including as set forth by U.S. Published Patent Application No.2003/0170617 filed Jan. 28, 2003.

In some embodiments, biomolecules including one or more polynucleotidesor polypeptides are spatially encoded. In some embodiments, thepolynucleotide can include a single-stranded or double-strandedpolynucleotide. In some embodiments, the polypeptide can include anenzyme, antigen, hormone or antibody. In some embodiments, the one ormore biomolecules can include RNA, mRNA, cDNA, DNA, genomic DNA,microRNA, long noncoding RNA, ribosomal RNA, transfer RNA, chloroplastDNA, mitochondrial DNA, or other nucleic acids.

It will be readily apparent to one of ordinary skill in the art that theembodiments and implementations are not necessarily inclusive orexclusive of each other and may be combined in any manner that isnon-conflicting and otherwise possible, whether they be presented inassociation with a same, or a different, embodiment or implementation.The description of one embodiment or implementation is not intended tobe limiting with respect to other embodiments and/or implementations.Also, any one or more function, step, operation, or technique describedelsewhere in this specification may, in alternative implementations, becombined with any one or more function, step, operation, or techniquedescribed in the summary. Thus, the above embodiment and implementationsare illustrative rather than limiting.

Referring to FIG. 1, the Single Cell Spatial Analysis System 100 acceptsspecimens 301 and processes one or more microsamples 125 to encode thephysical location of the microsample 125 within the specimen 301 toproduce spatially encoded single cells 1000 in microdrops, e.g.,nanodroplets or boluses, by adding a marker such as a DNA barcode thatencodes for the location of the microsample 125. Known markers are addedin known order to ordered microsamples 125 to encode the spatialposition. Analysis method 1100, such as DNA sequencing, massspectrometry, or other analytical methods, generates single cellanalytical data 1600, such as DNA sequence or proteomics, and alsodecodes the encoded spatial information to produce decoded single cellspatial information 1500. Multiple sample preparation and analyticalmethods can be used on a specimen. In some aspects where thebiomolecules are nucleic acids, the downstream processes can includewithout limitation, nucleic acid sequencing, targeted resequencing,genotyping analysis, mutation analysis, copy number variationassessment, allele frequency assessment, plasmid construction, cloning,and the like. The decoded single cell spatial information 1500identifies the spatial position 130 where microsample 125 originated inspecimen 301. The single cell analytical data 1600 is analyzed by singlecell spatial analysis 2000 software to produce single cell spatialinformation 3000 which has the analytical data and associated physicalposition information of the microsample 125.

Referring to FIG. 2, the Single Cell Spatial Analysis System 100 is insome embodiments an automated sample preparation system that collectscells from areas of tissue, biofilms, and other matrices containingbiological material and generates spatially-encoded samples for analysisof single cells 1000 and groups of cells from the same subregion. TheSingle Cell Spatial Analysis System 100 can have pushbutton operationfor either specialists or non-specialists to generate samples withspatial information encoded from specimens for medical, health, lifescience research, and other applications.

The Single Cell Spatial Analysis System 100 in one embodiment has aSpatial Subsystem 200 that inputs specimens and outputs samples forspatially encoded single cells 1000. The Spatial Preparation Subsystem200 can accommodate many different types of specimens, comprised offresh and snap-frozen tissue in the form of microtome slices (cryo,laser or vibrating); bulk material obtained by surgical excision,biopsies, fine needle aspirates; samples from surfaces, and othermatrices. The specimens are positioned onto the sampling stage.Subregions 150 will then be addressed automatically by a multifunctionalhead 330 to transfer subregions 150 of microsamples 125 in 2-D spatialorder to generate single cells and process them into cDNA or DNA with aspatial nucleic acid barcode, or other spatial markers added, or otheranalytes, e.g., proteins, metabolites, enzymes, with isotopes or otherspatial markers added. The next physical layer of the specimen 301subregion of interest can then be collected and analyzed to generate asecond 2-D spatial patterns of DNA sequence, RNA expression, proteinexpression, protein activity, RNA activity, lipid composition andabundance, carbohydrate composition and abundance, and metabolites aswell as any other biological components. The third physical layer canthen be collected and analyzed. The physical layers can then be orientedto produce a three dimensional pattern.

The basic elements of the Single Cell Spatial Analysis System 100 can beconfigured in multiple ways depending on the specimen(s) and analytes tobe analyzed. In the following examples, a few of the numerousconfigurations are described in detail but in no way is the inventionlimited to these configurations as will be obvious to one skilled in theart. Nonetheless in many configurations, there are many common elements,in particular adding a barcode or other exogenous material to themicrosample 125, or to single cells or groups of single cells from themicrosample 125 to encode where physically from the specimen themicrosample 125 originated. Once the single cells or groups of cellshave been analyzed, the barcode can be decoded and the physical positionof the microsample 125 in the 3-D structure of the specimen used tounderstand the composition, function, cell type, activity, genetics,physiology, interconnections, and other attributes of the specimen withsingle cell spatial information 3000.

In a preferred embodiment, referring to FIG. 3, for nucleic acidanalysis, the Single Cell Spatial Analysis System 100 has twosubsystems, the Spatial Preparation Subsystem 200 and the SpatialLibrarian Subsystem 500.

The Spatial Preparation Subsystem 200 can in turn be composed of twomodules, the Spatial Sampler module 300 and the Spatial Encoder 400, asshown in FIG. 4. In a preferred embodiment, Spatial Sampler module 300(i) releases cells from tissue, (ii) collects, and (iii) transferssingle cells or groups of cells with defined spatial relationships in aknown order into a single fluidic or microfluidic process stream, oronto a surface such as a flow cell, or into a two-dimensional array ofwells, or other ordered or unordered processes. In a preferredembodiment, the output of the Spatial Sampler 300 is single cells in amicrofluidic flow in a known order. Spatial Encoder module 400 addsspatial barcodes to the sample and outputs the single cells with spatialbarcodes. FIG. 4 shows Spatial Encoder module 400 configured for DNAsequencing.

A. Spatial Sampler Description

Referring to FIG. 5, in a preferred embodiment, configured for nucleicacid analysis, specimen 301 is placed in a specimen holder 310 which isinserted into the Spatial Sampler module 300 by loading mechanism 305.Specimen holder 310 may be temperature controlled. The loading mechanismcan have a mechanical slide, stage, pneumatic actuator, or othermechanism, that accepts specimen holder 310 and moves it into the SingleCell Analysis System 100, either automated or manually, into a fixedposition in the Spatial Sampler module 300 or other mechanism. SpatialSampler module 300 can have a three axis stage 320 that moves amultifunctional head in the x, y and z directions 330 to collectmicrosamples 125 in a spatially defined manner from specimen 301 atcapture position 345 and dispenses the collected microsamples 125 atdispense position 350 into input device 360.

In some embodiments the fluidics of the Single Cell Spatial AnalysisSystem 100 are incorporated onto cartridge(s) 4000. In some embodimentsof the Single Cell Spatial Analysis System 100, the valves for thesubsystems are microvalves, which in some embodiments are created inmicrochips with microchannels. Microvalves are well know to thoseskilled in the art. Microvalves can be actuated by, for example,mechanical force, pneumatic pressure, electrostatic force, piezoelectricforce, or thermal expansion force. They may be have internal or externalactuators. Pneumatic valves include, for example, diaphragm valves thatemploy a flexible membrane of the pneumatic pressure or vacuum to closeor open a fluid channel. Electrostatic valves may include, for example,a polysilicon membrane or a polyimide cantilever that is operable tocover a hole formed in a substrate. Piezoelectric valves may includeexternal (or internal) piezoelectric disks that expand against a valveactuator. Thermal expansion valves may include a sealed pressure chamberbounded by a diaphragm. Heating the chamber causes the diaphragm toexpand against a valve seat.

Microvalves can comprise mechanical (thermopneumatic, pneumatic, andshape memory alloy), non-mechanical (hydrogel, sol-gel, paraffin, andice), and external (modular built-in, pneumatic, and non-pneumatic)microvalves (as described in C. Zhang, D. Xing, and Y. Li., Micropumps,microvalves, and micromixers within PCR microfluidic chips: Advances andtrends. Biotechnology Advances. Volume 25, Issue 5, September-October2007, Pages 483-514; Diaz-Gonzalez M., C. Fernandez-Sanchez, and A.Baldi A. Multiple actuation microvalves in wax microfluidics. Lab Chip.2016 Oct. 5; 16(20):3969-3976.; Kim J., Stockton A M, Jensen E C,Mathies R A. Pneumatically actuated microvalve circuits for programmableautomation of chemical and biochemical analysis. Lab Chip. 2016 Mar. 7;16(5):812-9. doi: 10.1039/c51c01397f; Samad M F, Kouzani A Z. Design andanalysis of a low actuation voltage electrowetting-on-dielectricmicrovalve for drug delivery applications. Conf Proc IEEE Eng Med BiolSoc. 2014; 2014:4423-6. doi: 10.1109/EMBC.2014.6944605.; Samad M F,Kouzani A Z. Design and analysis of a low actuation voltageelectrowetting-on-dielectric microvalve for drug delivery applications.Conf Proc IEEE Eng Med Biol Soc. 2014; 2014:4423-6. doi:10.1109/EMBC.2014.6944605.; Lee E, Lee H, Yoo S I, Yoon J.Photothermally triggered fast responding hydrogels incorporating ahydrophobic moiety for light-controlled microvalves. ACS Appl MaterInterfaces. 2014 Oct. 8; 6(19):16949-55. doi: 10.1021/am504502y. Epub2014 Sep. 25.; Liu X, Li S. An electromagnetic microvalve for pneumaticcontrol of microfluidic systems. J Lab Autom. 2014 October;19(5):444-53. doi: 10.1177/2211068214531760. Epub 2014 Apr. 17; Desai AV, Tice J D, Apblett C A, Kenis P J. Design considerations forelectrostatic microvalves with applications inpoly(dimethylsiloxane)-based microfluidics. Lab Chip. 2012 Mar. 21;12(6):1078-88. doi: 10.1039/c21c21133e. Epub 2012 Feb. 3.; Kim J, KangM, Jensen E C, Mathies R A Lifting gate polydimethylsiloxane microvalvesand pumps for microfluidic control. Anal Chem. 2012 Feb. 21;84(4):2067-71. doi: 10.1021/ac202934x. Epub 2012 Feb. 1; Lai H, Folch A.Design and dynamic characterization of “single-stroke” peristaltic PDMSmicropumps. Lab Chip. 2011 Jan. 21; 11(2):336-42. doi:10.1039/c01c00023j. Epub 2010 Oct. 19) In some embodiments, cartridges4000 are used with functionality from a group of reagents, valves ormicrovalves, microchannels, syringe pumps, optical devices, integratedelectronics for control of cartridge 4000 functions, and lot tracking.In some embodiments microchips are used as parts of instrument orcartridges 4000. The cartridges 4000 in some embodiments hold kits toperform the chemistries including all needed reagents, including spatialbarcodes attached to beads, emulsion oil, breaking solution, stains,library preparation chemistry, and other consumables.

The multifunctional head 330, supported by strut 340, can have multiplefunctions integrated into a single head as shown in FIG. 6 or multipleheads that are moved independently can be used. As shown in FIG. 6, themultifunctional head can have an optics head 331 that in a preferredembodiment has illumination fiber 332 surrounded by multiple collectionfibers 333. The illumination and collection fibers can have lenses tofocus the illumination beam(s), such as a laser, arc lamp, UV lamp, orother light source, and collect reflected, fluorescent, Raman,reflected, or other light for delivery to an optics analysis device viaoptical fiber bundle 338. Many other optical configurations may be usedor no optical capability may be used.

Multifunctional head 330 can have a dispense head 334 with one or moredispensers 335 that can apply one or more solutions to specimen 301. Thedispense head 334 may use piezoelectric pumps, inkjet technology,pressure driven flow through a nozzle, or other methods to dispenseliquids, gels, or gases from fluidics and pneumatic bundle 339 tospecimen 301 or subregions 150 of specimen 301.

Multifunctional head 330 can have a transfer head for extracting themicrosamples from a biological specimen and transferring them, e.g.,into a fluidic stream. The transfer head can comprise a transfermembrane 336 to transfer a layer of the specimen. Referring to FIG. 6, atransfer membrane 336 may be made of many different materials withcharacteristics that prevent cells passing through it but in preferredembodiments can be permeable to air or liquids, have low adhesion forcollected cells, and can be changed as needed. The transfer membrane maybe selected from many materials, including semipermeable membrances;filter papers comprising cellulose and cellulose derivatives, glassfiber filters, polytetrafluoroethylene (PTFE) filters, quartz fiberfilters and others; blotting membranes comprising membranes made ofnitrocellulose such as ProTran, polyvinylidene difluoride (PVDF) such asImmobilon® or Hybond, nylon, cellulose nitrate, polyestersulfone, tracketched polycarbonate, PVC, and others with pore sizes smaller than thecells of interest. The multifunctional head 330 may be able to applyvacuum, pressure, or fluids to the whole membrane through fluidic andpneumatic bundle 339 either to the complete surface of the membrane atthe bottom of the multifunctional head or to portions of the membrane.In some embodiments, portions of the membrane are individuallyaddressable subregions 337 to enable subregions of the specimen 301 tobe collected or dispensed individually. The transfer membrane 336 can bechanged through swapping out a disposable fixed membrane head, or byadvancing a roll of membrane to have unused membrane, or othermechanisms. In some embodiments, the transfer membrane may be reusedmultiple times and not changeable. In other embodiments, the transfermembrane 336 is simply cleaned by separate cleaning modules that operatein direct contact or non-contact mode utilizing various cleaningmechanisms including, but not limited to, mechanical brushes andchemical agents such as ethanol, detergents, water, buffers, and otherchemicals. The term transfer membrane 336 is used but is not intended tolimit the composition or material which can be a filter, membrane,surface, film, or other material.

The Single Cell Spatial Analysis System Spatial Sampler module 300 canfunction as follows in one embodiment. The multifunctional head 330 ismoved to the x, y directions to capture position 345. The optics head331 scans the specimen by the multifunctional head 330 moving to theappropriate z height and being moved in the x-y axis to scan specimen301. As needed, solutions can be dispensed onto specimen 301 or portionsof the specimen by dispense head 334. The solutions can be imagingreagents, such as fluorescently directly conjugated antibodies orsecondary antibodies, stains, fluorescent probes and dyes; imagingnanomaterials (including quantum dots and other nanoparticles), or othercontrast or straining reagents; or reagents to stimulate or treatspecimen 301 such as anticancer compounds, antibiotics, antivirals,energy sources, or other liquids, gels, or gases for the same or otherfunctions. In some embodiments, the information gathered by the opticsor other sensors is used to decide which subregions 150 of the specimenare to be sampled or to decide the next step, such as adding anotherreagent, scanning at a different wavelength, or another action. In someembodiments, after the application of a reagent, the transfer membrane336 portion of the multifunctional head 330 may be used to aspirateexcess reagent or all unbound reagents.

In a preferred embodiment, after optical scanning, the dispense head 334can use a dispenser 335 to apply a dissociation solution, such asLiberase™ DH Research Grade Roche or equivalent products or customformulations, in form of a spray, mist, or liquid to a region of the topmost layer of the tissue to dissociate intercellular adhesion betweencells and free the cells from the extracellular matrix. A dissociationsolution comprises at least one collegenase or protease that digestsextracellular matrix (F. E Dwulet and M. E. Smith, “Enzyme compositionfor tissue dissociation,” U.S. Pat. No. 5,952,215, Sep. 14, 1999). Thedissociation solutions to dissociate the tissue can be comprised ofcollagenases (e.g. collagenases type I, II, III, IV, and others),elastase, trypsin, papain, hyaluronidase, chymotrypsin, neutralprotease, clostripain, caseinase, neutral protease (Dispase®), DNAse,protease XIV or other enzymes. The dissociation solution can be appliedin one embodiment such that only the uppermost layers of cells aredissociated or deeper layer are dissociated depending on the amount andconcentration of the dissociation solution, time of contact,temperature, and other parameters.

Referring to FIG. 7a , after treatment to dissociate the tissue, samplesare collected from the specimen by moving multifunctional head 330 inthe x and y axes to the appropriate subregion 150 of specimen 301 andthen multifunctional head 330 is lowered in the z direction to contactspecimen 301 in specimen holder 310 with the transfer membrane 336.Cells from the top layer of the dissociated tissue are gently picked upby vacuum, contact, absorption, and other methods to air-permeable,cell-impermeable, transfer membrane 336 located at the bottom of themultifunctional head 330 for sample transfer; this can be similar tovacuum assisted blotting when vacuum is used. The transfer membrane 336limits the amount of transfer to preferably less than 20 um or less than100 μm of tissue thickness and minimizes contamination by replacing thesurface of the transfer membrane 336 with fresh membrane materialbetween sequential sampling steps or cleaning. In some embodiments, anarray of individually addressable vacuum/pressure or fluidic linesbehind the membrane can be used to selectively apply vacuum toindividually addressable subregions 337 of the membrane.

The multifunctional head 330 is then raised in the z direction, moved inthe x and y direction as needed to dispense position 350, and lowered tocontact the input device 360. FIG. 7b shows multifunctional head 330about to contact input device 360. Input device 360 may contact all ofthe transferred specimen 302 or a subregion 150 of the transfer membrane336. In a preferred embodiment, the input device 360 is an array ofmicrofluidic channels. It will be apparent to one skilled in the artthat the fluidics and microfluidics may be implemented in differentforms including capillaries, molded microchannels, embossedmicrochannels, etched microchannels, and many other formats in manymaterials including plastics, glass, silicon dioxide, metals, andothers. FIG. 7b shows a closeup of two ends of the input microchannels382 held in connectors 316 about to contact the material transferredfrom specimen 301.

FIG. 8a shows a bottom view of multifunctional head 330 that hastransferred parts of specimen 302 onto transfer membrane 336 aftercontact by input device 360. Subregion 150 is outlined as the portion ofthe transfer membrane 336 that will be transferred into input device 360in this example.

FIG. 8a shows 12 microsamples 125 from the spatial positions 130 (shownas black circles) have been transferred onto transfer membrane 336 andthen into input microchannels 382. The transfer into input microchannels382 may be any number of microsamples 125 at a time.

A 2×6 array of input microchannels 382 produces the pattern ofmicrosamples 125 from spatial positions 130 shown in FIG. 8a in oneinput step. The second input step can produce a pattern of 24microsamples 125 from spatial positions 130 that have been transferredas illustrated in FIG. 8b . Two more inputs steps would complete theinput of 48 microsamples from subregion 150. In a preferred embodiment,the whole surface of transfer membrane 336 is used to transfer cellseach time. In some embodiments the first pass of sampling microsamples125 from subregion 150 of transferred specimen 302 may be followed by asecond pass to transfer of cells from a second layer deep of transferredspecimen 302 into input device 360. In another embodiment, a differentsubregion 150 of transferred specimen 302 may be input in the secondtransfer. In another embodiment, after input of a set of microsamples125, the multifunctional head 330 can be moved to capture position 345and another sample from specimen 301 collected from the same ordifferent region of specimen 301 by transfer membrane 336; in someinstances, additional optic information is gathered, the specimentreated with reagents by dispensers 335, or other activities may beperformed before or after sampling specimen 301.

Input device 360 can adopt many different configurations. FIG. 8 showsthe microsamples transferred to input device 360 that are circular inshape, like a capillary or other round microchannel would produce. Theinside diameter (ID) can be matched to the size of the cells or materialto be sampled. Capillaries could be used with, for example, 5 μm m ID,or 20 μm ID, or 100 μm or 100s of μms to sample from different matrices.A 20 μm ID capillary can be used to sample mammalian tissue for singlecells and input a microsample 125 from 30 μm deep for example; thismicrosample 125 could contain a single or multiple cells. A 100 μm IDcapillary can be used to create microsamples 125 that could be 75 μmdeep, for example, and might contain 10s or more cells. Other shapesthan circular will enable sampling with different resultant patternssuch as rectangular, triangular, and other shapes.

The number of samples collected from transfer membrane 336 will dependon the number of input microchannels 382 in input device 360. In someembodiments it is a single input microchannel 382 and in otherembodiments two or more microchannels that may be arranged in one ormore rows and columns. In some embodiments input device 360 collectsmicrosamples 125 from the complete surface of transfer membrane 336simultaneously.

The pattern of transfer of microsamples 125 from transferred specimen302 is known and where in the subregion 150 input device 360 contactedtransferred specimens 302 is known, therefore the spatial position 130of where in the transferred specimen 302 each microsample 382 originatedis known; therefore the Single Cell Spatial Analysis System 100 can keeptrack of the order of each microsample 125 until it is encoded with amarker or barcode to spatially encoded single cells 1000.

After encoding, as described below in detail, the microsamples 125,cells, or groups of cells, can then be pooled if desired. After samplepreparation and analysis by an analytical method 1100, e.g., DNAsequencing, mass spectrometry, etc., the barcode is read out to produceDecoded Single Cell Spatial Data 1500. Decoding the barcode thendetermines from which microsample 125 each single cell orginated sinceknown barcodes were added to ordered microsamples 125 in known order.Since the spatial position 130 of each microsample 125 is known, thethree dimensional or two dimensional position of the single cells andgroups of cells in multiple microsamples is known. Single Cell SpatialAnalysis 2000 software analyzes the spatial position information withthe spatial position 130 of each microsample 125 with Single CellAnalytical Data 1600 to produce Single Cell Spatial Information 3000.

FIG. 9 shows one configuration of a single channel implementation of theinput device 360. In this embodiment, the input device 360 connects ofinput microchannel 382 through two three-way valves to one or moresyringe pumps. The end of input microchannel 382 contacts the transfermembrane 336 through an optional filter 381 such as a 50 μm stainlesssteel mesh to break up any clumps of cells and remove debri. Syringepump 365 pulls reagents from reagent rail 366 using three-way valve 364and fluidic channel 367 from one of the reagents, 369, 371, 373, and 375with selection controlled by valves 368, 370, 372, and 374.

To input a microsample 125 into the end of input device 382, in oneembodiment syringe pump 365 pulls buffer, e.g., 100 nL of PBS buffer,from buffer reservoir 369 through fluidic channel 367 by opening buffervalve 368 and three-way valve 364; a sample loop can be placed inbetween syringe pump 365 and three-way valve 364 or other locations asneeded. Three way valve 364 can be replaced with valves such as 3 way ormore way valves. After switching three-way valve 364, syringe pump 365then pushes the buffer through three-way valve 364 through definingmicrochannel 363, three-way valve 362, input microchannel 361, the endof input microchannel 382 and through filter 381 to contact the portionof the transferred specimen 302 on transfer membrane 336. Pressure orliquid can be supplied through fluidic and pneumatic bundle 339 toindividually addressable subregions 337 of the areas that match inputdevice 360 to help elute microsamples 125 off of the transfer membrane336 and into input device 360 as needed.

After changing the state of three-way valve 364, syringe pump 365 canpull the buffer and the transferred cells from transferred specimen 302,and moving it into input device 360, through filter 381, end of inputmicrochannel 382, input microchannel 361, three-way valve 362, and intodefining microchannel 363 which is flanked by three way valves 364 and362 to create a microsample 125 in the fluidic stream. Three way valve362 is then changed to connect defining microchannel 363 to connectingmicrochannel 376 and flow channel 380 and syringe pump 365 used to pushthe microsample 125 from defining microchannel 363 through three wayvalve 362 and connecting microchannel 376 and flow channel 380. Valve379 may be closed to direct the flow in the direction of the arrow,towards the Sampler Encoder module 400 or other downstream processing.

Flow movement in flow channel 380 can also be accomplished using flowsyringe pump 390 accessing flow reagent rail 391 through flow three wayvalve 393 to push fluids or gases to move the bolus(es) entrained inflow channel 380.

One or more optional bolus detector 396 can detect the edges of bolusesusing optical measurement, conductance, or other methods well known toone skilled in the art. The bolus detectors can be used to align theadditional encoding beads downstream as needed.

The length of the bolus containing the microsample 125 can be defined bythe volume in defining microchannel 363 which is flanked by three wayvalves 364 and 362, and the length and cross-sectional area of definingmicrochannel 363. In one preferred embodiment, the volume of the bolusis 100 nL, but volumes can range from subnanoliters to 10s ofmicroliters or larger.

The bolus can be flanked by many materials including air, immisciblefluids (such as Fluorinert®; mineral oil; silicone-based oil;fluorinated oils; Droplet Generation Oil (Biorad, #1863005); emulsionoil (Life Technologies, Part No. 4469000); 73% Tegosoft DEC(Diethylhexyl Carbonate), 20% mineral oil, and 7% ABIL WE; 0.12% Span(v/v), 0.00325% Tween 80 (v/v), 0.0000125% Triton X-100 (v/v) in mineraloil; mineral oil containing 2% (v/v) ABIL EM 90 and 0.05% (v/v) TritonX-100 (Tanaka H, Yamamoto S, Nakamura A, Nakashoji Y, Okura N, NakamotoN, Tsukagoshi K, Hashimoto M. Hands-off preparation of monodisperseemulsion droplets using a poly(dimethylsiloxane) microfluidic chip fordroplet digital PCR. Anal Chem. 2015 Apr. 21; 87(8):4134-43. doi:10.1021/ac503169h. Epub 2015 Apr. 7.) and other formulations) fluidswith crowding agents such as polyethylene glycol that limit diffusionand interaction of the cells, fluids with reaction components, and manyothers. These materials can help define the placement of the bolus inthe downstream flow, prevent mixing or dilution of microsample 125,provide reactants, or other functions. As used herein, the term“immiscible fluid” refers to a fluid that is immiscible with a solutioncontaining a microsample.

FIG. 10 shows an embodiment with the input device(s) 360 having twochannels of input, through end of input microchannels 382 and 392. Thetwo or more channels can be operated by two or more syringes 365 and 395as shown in FIG. 10 or the microchannels can be ganged together tooperate from a single syringe in parallel. Similarly the two channelsmay have separate reagent rails 366 and 387 or a single reagent rail.Multiple channels of input may have a single or reagent rails, forexample, a bank of 16 input devices 360 could have two reagent rails,each of which services eight input channels. In some instances,different sets of reagents may be used in the different reagent rails,e.g., RNA sequencing preparation in one reagent rail and DNA targetedsequencing reagents in a second reagent rail. Many additionalconfigurations are possible. It is within the scope of the presentdisclosure that one reagent such as 371 contain a dissociation solutionto further loosen cellular attachments to extracellular matrix material.The dissociation solution or other solutions could be applied throughthe fluidics to a microregion of the sample on transfer membrane 336before transfer of the microsample 125 or be used as the fluid totransport the microsamples to continue to dissociate any clumps. Toseparate boluses downstream flow microchannel 380 and outputmicrochannel 386, one reagent can hold an immiscible fluid such asFluorinert, mineral oil, Droplet Generation Oil (Biorad, #1863005) orother fluids. A cleaning reagent to remove debri, disinfect, flush,andother functions can be used in reagent 375. The number of reagents maybe as low as one and be as large as needed for the application withoutlimitation.

In some embodiments the reagents are in syringes operated by syringepumps. In another embodiment, the reagents are in pouches, tubes, wells,or other containers and are moved by a pump or micropump such as apiezopump, e.g. a Bartels mp6, Dolomite Piezoelectric Pump 3200138, orothers. In some embodiments, the reagents and other components of theSingle Cell Spatial Analysis System 100 may be in cartridges 4000 thatare readily changed by non-specialists.

When transfer membrane 336 is moved against the input device 360, if twoor more end of input microchannels 382 and 392 are used, they may bedirectly adjacent or with known spacing. In a preferred embodiment theends are adjacent. When the input microchannels are not adjacent, thetransfer membrane 336 can be moved against the input device 360 formultiple samplings of the transfer membrane 336. For example, if theinput device has microchannels of 20 μm ID and spacing betweenmicrochannels of 200 μm, then the multifunction head 330 could move tentimes to pull in samples from the complete subregion 150 if it islinear. Similarly, multifunction head 330 could raster or use otherpatterns to insure that all of the transfer membrane 336 is sampled whendesired.

Microsamples 125 for multiple input microchannels may be processed intodefining microchannels 363 and 394 in parallel or independently.Similarly, to move the microsamples 125 downstream of flow channels 378and 380 to be output into microchannel 386, three way valves 362 and 399are changed to connect defining microchannels 363 and 394 to connectingmicrochannels 376 and 383 to flow channels 380 and output microchannel386. Valve 379 may be closed to direct the flow in the direction of thearrow, leading to the Sampler Encoder module 400 or other downstreamprocessing. In one embodiment, the microsample 125 in definingmicrochannel 394 is pushed by syringe pump 395 into output microchannel386 and then the upstream microsample 125 in defining microchannel 363is pushed by syringe pump 365 into flow channel 380 to place the twosamples in a known order, separated by the volumes and initial materialin connecting microchannel 376 and flow channel 380. In anotherimplemention the microsamples 125 may be moved simultaneously.

To dispense microsamples 125 into the microfluidic stream in a knownorder, the multifunctional head or other device moves the transfermembrane 336 to a dispense position where released cells are pulled inthe order of the original 2-D spatial position into end of inputmicrochannels 382 and 392 on input device 360, driven initially bysyringe pumps 365 and 395. For example, with an array of a column ofmicrochannels on the input device, the top row could be input column bycolumn. Boluses of air, immiscible fluids, or other gases or liquids canbe introduced to separate microsamples 125 as needed. Cells are alignedin order and output in a single fluidic or microfluidic stream into theSpatial Encoder module 400.

i. One Channel Spatial Sampler Example.

In one implementation, a transfer device with a membrance with a singlemicrochannel can be implemented using a single capillary, e.g., 20 μm IDcapillary (Polymicro Technologies), which in a prefered embodiment isepoxied in a fiberoptic-like capillary (FROLC) connector with a membraneattached to one end, held in a moveable fixture; the other end of thecapillary is attached through a three way valve using a FROLC connectorto defining microchannel 363 of 100 nL to reagent rail 366 and syringepump 365 as a vacuum and pressure source. The single 20 μm ID capillarymay be able to sample about 1 nL from specimen 301.

The FROLC connectors, shown in FIG. 11, are true zero dead volumeconnectors and can join two or more capillaries to one, and be used asmultiway microvalves. (Jovanovich, S. B. et. al. Capillary valve,connector, and router. Feb. 20, 2001. U.S. Pat. No. 6,190,616Jovanovich; S. B. et. al. Method of merging chemical reactants incapillary tubes. Apr. 22, 2003. U.S. Pat. No. 6,551,839; Jovanovich, S.,I. Blaga, and R. McIntosh. Integrated system with modular microfluidiccomponents. U.S. Pat. No. 7,244,961. Jul. 17, 2007.) are incorporated byreference and their teachings which describe the FROLCs and details ofFROLC connectors, including their use as multiway valves, routers, andother functions including microfluidic circuits to perform flowthroughreactions and flow cells with internally reflecting surfaces. FROLCs canbe made by using a fiberoptic FC-, or other fiberoptic connectors 311with cleaved capillaries plugged with wax or other materials, and theninserted into face 312, epoxy filled, polished to a dome like a fiberoptic connector, and the wax removed. A spring in the connector providescompression to seal capillaries for liquid flow at >9,600 psi. Rotationof the connector can change the orientation of, for example, threecapillaries to rout sample streams into any of three output capillaries.FROLC connectors can also be made from other designs than fiberopticconnectors in a matter that allows leak-free connection between one ormore capillaries or microchannels. A FROLC connector is shown in FIG.11a with three capillaries 313, 314, and 315, that are visible in FIG.11 b.

Membranes such as transfer membrane 336 or filters on input devices,such as a 20-40 um range filters or other materials can be mounted tothe FC stainless surface over the end of the capillary in the FROLC(FIG. 11c ). A collar to simplify mounting the membrane can be made with3-D printing or other methods. Collection and dispensing requirements ofdifferent membranes can be evaluated for the ability to ‘grab’ the toplayer from loosened tissue and to dispense into the input device 360.

Membrane filters can be from commercial sources with differentcharacteristic, such as material, coating, and pore size. The amount ofvacuum to pickup and hold, pressure to dispense, efficiency in pickupand delivery, and the depth of tissue picked up can be optimized fordifferent membranes and specimens; the part of the FC connector withoutthe capillary can be used as a control region when evaluating transfermembrane 336 candidates. Membranes can be examined under brightfieldand, after fluorescent cell stains, with fluorescence microscopy fortesting of capture and dispensing. Selected samples can be assayed withreal-time PCR to develop the process and the requirements for repeatedlycollecting cells onto a surface and then dispensing using a singlecapillary, and provide first insight into repeated sampling to generate3-D spatial information.

To input a microsample 125 from transfer membrane 336 in a singlechannel implementation using a capillary held in a FROLC connector 311for the input device 360, a microsample 125 can be pulled into a singlechannel with a capillary (e.g., 75 μm ID) with FROLC connectors on bothends. One end will contact the transfer membrane and might optionallyhave a filter or mesh on its outer surface to breakup cell clumps; theother end is a FROLC three way valve that is in turn attached to thedefining microchannel 363 which can have FROLC at both ends with theFROLCS used as three way microvalves to connect at the upstream end tothe input device end 382 and to a capillary as the connectingmicrochannel 376 and the other end of defining microchannel 363 througha FROLC three way valve to a syringe pump 365 and a reagent rail 366.Buffer, ˜100 nL, will be delivered to the tip of the capillary andmanually contacted to the transfer membrane area of the multifunctionalhead 330. Transfer protocols can be optimized for requirements forpulling samples into the input device, timing, and fluidic process.

The defining microchannel 363 can readily be a volume such as 100 nL, orlarger or smaller. A 100 nL is an approximately 7 cm long bolus in a 40μm ID capillary or 2.5 cm long bolus in a 75 μm ID capillary—sufficientlength to be defined between two FROLC or other microvalves. If needed,the 100 nL sample can have immiscible fluid added to create a largervolume to manipulate. The ability to input a sample and then rout intothe microfluidic flow can be tested with fluorescent beads and then cellsuspensions. Multiple samples of alternating types (i.e., rat cellsfollowed by murine cells) can be input and output to the fluidics streamto measure cross contamination rates. Cleaning solutions in reagent 375can be used. In a single channel example, the connecting microchannel376 can be used as the output line for the Spatial Sample module(microchannels 378 and 380, and valve 379 are not used). In addition tousing a capillary, in a single channel implementation, it is within thescope of the present disclosure to use microchannel(s) that may beinjection molded, milled, or otherwise made and covered with a materialsuch as a heat-sealed membrane.

ii. Multi-Channel Spatial Sampler Example.

If neither pressure is needed to dispense from the membrane nor iscontinued vacuum needed to hold samples onto the membrane, the sampletransfer portion of the multifunctional head 330 simplifies to atransfer membrane 336 with vacuum control. If dispensing requirespressure sequentially applied to individual subregions of the membrane,then to achieve high throughput the transfer device can be an arraydevice that input collects and dispenses multiple samples at a time intoa ‘mirrored’ input format, i.e., if the transfer device used 8×12channels, the input device 360 would use 8×12 channels.

FIG. 12 shows 12 capillaries 317 arrayed in a linear FROLC connector316. For transfer, in one embodiment, the transfer region of themultifunctional head will have a membrane in front of the 12 capillariesto collect samples, each capillary individually addressable to deliverpressure to dispense the 12 microsamples 125 if needed. The input device360 can be be another linear array of 12 capillaries 317 in inear FROLCconnector 316, covered with a 30 μm filter, connected to a gangedsyringe pump an array of 12 fluidic circuits. In other embodiments, thetransfer region of the multifunctional head does not have a membrane infront of the 12 capillaries to collect samples.

a. Spatial Encoder Description.

The Single Cell Spatial Analysis System Spatial Encoder module 400inputs the ordered microsamples 125 which may contain single cells orgroups of cells from the Spatial Sampler module 300 using the SpatialSampler output such as output microchannel 386 as an input to theSpatial Encoder module 400. The spatial encoder subsystem can place themicrosamples in a sequential ordered arrangement in a fluidic stream.This sequential arrangement is referred to as a train, and microsamplesin a train are said to be entrained. In a preferred embodiment fornucleic acid encoding, adds beads with known barcodes to correlate withthe original spatial information into boluses, creates microdrops,(nanodroplets in some implementations, or boluses in otherimplementations), preferably with one or less cells per nanodroplet. Abolus is typically elongate in shape, while a nandroplet is typicallyspherical. A bolus typically has a volume of at least 3 microliters. Ananodrop typically has a volume of no more than 3 microliters, e.g.,about 1.5 microliters. In a preferred embodiment, the Spatial Encodermodule 400 outputs single cells with spatial barcodes in nanodroplets orboluses to the Spatial Librarian Subsystem 500.

One embodiment of spatial barcoding is to use beads witholigonucleotides with spatial barcodes 680 is illustrated in FIG. 13a .The beads with oligonucleotides with spatial barcodes 680 can beparamagnetic beads, agarose beads, or others, and have surface chemistryoptimized for the nucleic acid capture and subsequent chemistries.Oligonucleotides with spatial barcodes 601 can be generated by synthesisusing standard commercially available phosphoramide or other technology.In one embodiment, the oligonucleotide has a cleavable linker 602,attached to an amplification primer 604 with fluorescent label 603, asequencing primer 605, barcode region 606, and capture region 610. Thebarcode region is comprised of a spatial barcode 607, cellular barcode608, and molecular barcode 609. In one embodiment the spatial barcode607 can be 5 nucleotides long to provide 1,024 barcodes for spatialresolution. Cellular barcodes 608 can be 6 nucleotides, or otherlengths, and synthesized by split-pool synthesis, and molecular barcode609 can be 8 nucleotides synthesized by degenerate synthesis; thecellular barcodes 608 only need to identify cells from within a singlemicrosample 125 since each microsample is encoded with a spatialbarcode. In a preferred embodiment, each spatial barcode 607 is uniquefor each set of microsamples 125 that are analyzed together. In otherembodiments, spatial barcode 607 can be shared between microsamples 125and then resolved bioinformatically using cellular barcode 608 to sortand cluster by cells to resolve spatial barcode ambiguities. Spatial,cellular, and molecular barcodes can be of different lengths or indifferent orders, or dispersed among other elements of theoligonucleotide with spatial barcode 50 without limitation.

Another embodiment of spatial barcoding is to attach oligonucleotideswith spatial barcodes 601 to surfaces 660 as illustrated in FIG. 13b ,or flowcells 670. The surface 660 then can be used in similar ways tothe beads. It will be obvious to one skilled in the art that surface 660could be assembled into a flow cell.

The oligonucleotides, such as amino-modified oligonucleotides, can beinitially attached to commercially available paramagnetic beads 630 bycovalent crosslinking and may include a cleavable linker bond (Ju. J.et. al. U.S. Pat. No. 9,133,511. Sep. 15, 2015.), (Knapp D. C. et. al.Bioconjug Chem. 2010; 21(6):1043-55.),(https://www.clickchemistrytools.com/products/click_chemistry_toolbox),(Olejnik J. et. al. Nucleic Acids Res. 1996; 24(2):361-6.). Fluorescentprobes can be attached to the oligonucleotide distal from the bead andcleavable bond or alternatively fluorescent nucleic acid base analogscan be used such as 2-Aminopurine (Wilhelmsson, Quarterley Reviews ofBiophysics, 43, 2, 2010, 159-183). The cleavage of labeledoligonucleotides can be used for assay development since theoligonucleotide can be analyzed by fragment sizing on CE with thefluorescent tag to give the distribution of sizes to assess libraryquality.

The hardware and software of the Spatial Encoder module 400 must delivera set of beads with a unique, known, spatial barcode 607 to eachmicrosample 125. Referring to FIG. 14, the Spatial Encoding fluidicdelivery can use a spatial barcode reagent rail 401 to access beads eachwith a single spatial barcode, for example 403, 405, 407, and 409,controlled by spatial barcode reagent rail valves 402, 404, 406, and 408respectively, to deliver reagents through by spatial barcode fluidicchannel 410 to spatial barcode syringe pump 412. It is within the scopethat the spatially barcoded beads will scale to 1,024 or greater numberof spatial barcodes per run for the Single Cell Spatial Analysis System.Spatial barcode three way valve 411 and spatial barcode reagent rail 401can be implemented with many other variations, including having spatialbarcode reagent rail valves 402, 404, 406, and 408 each being multiwayvalves, such as 8-way valves that in turn access 8 sets of spatialbarcode reagents, or additional valves be added to the reagent rail. Themultiway valves can be FROLC or other microvalves, molded microfluidicvalves, valves on microchips, or other embodiments. An alternativeapproach is an ordered series of boluses of beads with different spatialcodes that are merged with the microsample 125 boluses in known order.

As microsamples 125 are moved from Spatial Sampler output, such asmicrochannel 386, reagent rail syringe pump 412 and spatial barcodereagent rail 401 can select a reagent of singly spatially barcodedbeads, such as beads all with a single spatial barcode 403, and deliversa bolus of beads 403 through spatial barcode connecting channel 413 tospatial encoder junction 414 to merge with the microsample 125 inspatial encoder microfluidic device 420. Optical, conductance, or othersensors can be incorporated as needed to detect the microsample 125 inthe bolus and coordinate the addition of the spatially barcoded beads tothe bolus.

The bolus then passes through spatial encoder microchannel 417 to nozzle429 where an immiscible fluid such as Fluorinert, Droplet Generation Oil(Biorad, #1863005), or other solutions can be added by nanodropletgeneration syringe pumps 425 and 415 to the bolus to producenanodroplets, preferably 1.5 nL, and sent down spatial encoder outputmicrochannel 430 as output from the Spatial Encoder. Nanodropletgeneration syringe pumps 415 and 425 can also be combined into onesyringe pump that has two microchannels 416 and 426 that split in twofrom nanodroplet generation syringe pump output to join the microsample125 with barcoded bead bolus from either side to produce nanodroplets,eliminating the need for a second nanodroplet generation syringe pump.Nozzle designs and circuits are incorporated by reference (Macosko E. Z.et. al. Cell. 2015; 161(5):1202-14.) (Klein A. M. et. al. Cell. 2015;161(5):1187-201.) (Geng T. et. al. Anal Chem. 2014; 86(1):703-12).

In an alternative embodiment, the microsample 125 bolus with the addedspatially barcoded beads are processed as a bolus without the formationof nanodroplets. In this approach, the bolus may be preferably less than5 nL, or 10 nL, or 25 nL, or 100 nL, or 250 nL,and 10,000 microsamples125 may be less than 2.5 mL.

In one embodiment, single channel fluidics are used. Referring to FIG.15, a bolus of, for example, 100 nL of beads with one spatial code areadded in junction 414 to the, for example, 100 nL of microsample 125 inoutput microchannel 386 and lysis and/or reaction mixtures, such aslysis/reverse transcriptase mix, added separately through spatialencoder reagent syringe pump 418 and reagent connecting microchannel419.

Monodispersed nanodroplets from single cells with spatially coded beadswith lysis and/or reaction mixtures, e.g., lysis/RT mix for RNA-Seq,lysis/restriction mix for DNA sequencing, are then be produced using anozzle 429 (Macosko E. Z. et. al. Cell. 2015; 161(5):1202-14.) (Klein A.M. et. al. Cell. 2015; 161(5):1187-201.) (Geng T. et. al. Anal Chem.2014; 86(1):703-12.) and output through spatial encoder outputmicrochannel 430. As needed, the geometry and flow rates can be alteredto adjust size and flow rates to produce a Poisson distribution ofsingle cells with each nanodroplet preferably having a spatiallybarcoded bead. In other embodiments, the bolus from the Spatial Samplermodule 300 is physically separated by structures, volumes, or surfaces,for example, by placing the bolus into a microtiter or smaller well ortube. The Spatial Sampler module 300 output can be used to be physicallydispersed onto the surface of a material comprised of agar, membranes,arrays of beads, microscope slides, flow cells, and others. The physicaldispersion can be by moving the surface under a capillary or other flow,by printing with a microarray pen, by piezospraying, electrowetting,microfluidics, or other methods. The physically separated microsample125 bolus can be dispersed such that, for example on the surface ofagar, all cells are far enough apart to be processed as single cells.

In one embodiment, spatial encoder reagent syringe pump 418 adds a lowmelting temperature agarose to encapsule the nanodroplet with heatedliquified agarose (Geng T. et. al. Anal Chem. 2014; 86(1):703-12.)during the formation of nanodroplets. Once cooled, the agarose can beused as a barrier permeable to low molecular weight components, such asreaction components, but not to high molecular weight components such asnucleic acids when it is cooled. The use of agarose to encapsulate thereactions enables multiple sequential reactions or manipulations in arow to be performed in the nanodroplet.

b. Spatial Librarian Subsystem Description

In one embodiment, the Spatial Librarian Subsystem 500 receives theoutput of the Spatial Preparation Subsystem 200 and can process themicrosamples 125 including single cells and groups of cells from asingle spatial position 130 of the specimen with a spatial barcodeadded. The Spatial Librarian Subsystem 500 can perform enzymaticreactions, chemical reactions, and purifications, quality control, andother functions.

Referring to FIG. 16, in one embodiment, the output from the SpatialEncoder module 400, e.g., output microchannel 430, is input into theSpatial Librarian Subsystem 500 to reaction device 520. In oneembodiment, the microdrops (e.g., nanodroplets or boluses) are pooled inreaction chamber 523 and reagents added from spatial librarian reagentrail 501 accessing spatial librarian reagents 503, 505, 507, and 509through spatial librarian valves 502, 504, 506, and 508 respectively toallow access through spatial librarian reagent rail connectingmicrochannel 510 with spatial librarian syringe pump 512 and spatiallibrarian three-way valve 511. Reagents can be added to reaction chamber523 to mix with pooled microdroplets. In other embodiments, reactiondevice 520 is a flowthrough system. Waste can be flowed into waste line526 as needed. Temperature can be controlled including thermal cyclingby temperature control device 521 which can include sensors and heaterswell known to one skilled in the art including resistive heating,infrared heating, flowing air or water, and other methods. Optionaloptic device 522 can monitor reactions such as quantitative polymerasechain reaction (qPCR), reverse transcriptase-PCR, Raman spectroscopy, orother optical measurements including fiberoptic delivery and collectionof light from lasers, filters, imaging devices such as CCD and CMOS, andother optical methods. Quality control device 525 can assess the qualityof the processing integrated devices such as capillary electrophoresiswith laser induced fluorescence to determine the amount and the sizedistribution of fragments in the library, mass spectrometry, Raman,electrochemistry, or other devices.

When needed, the spatial librarian can break or create emulsions byaccessing reagents on spatial librarian reagent rail 501. To break anemulsion, many standard methods can be used including adding isobutanol(“Idiot-proof emulsion PCR”, Lab Times, 1-2011, p50); isopropanol anddetergent buffer (10 mM Tris pH 7.5, 1 mM EDTA pH 8.0, 100 mM NaCl, 1%(v/v) Triton X-100, 1% (w/v) SDS); water-saturated diethyl ether; saltincluding sodium pyrophosphate; or demulsifiers such as Dow Corning® DM4Demulsifier or others can be added. In an alternative approach, anorganic extraction solvent, selected from the group consisting of:butanol, octanol, hexanol and chloroform,is added to an aqueous phase ofa breaking solution includes sodium dodecyl sulfate (SDS) or phosphatebuffered saline (PBS), further including an inorganic salt in theaqueous phase prior to forming the breaking solution wherein theinorganic salt is selected from the group consisting of potassiumchloride, potassium acetate, sodium chloride, sodium acetate, lithiumchloride, lithium acetate, Na2SO4, potassium carbonate, ammoniumsulfate, and ammonium acetate (Jeffrey Sabina, Ilya Zlatkovsky, RachelKasinskas, “Methods and kits for breaking emulsions” WO 2012138926 A1,Published Oct. 11, 2012) or. The solution can be allowed to separate bygravity and then the bottom phase collected with spatial librariansyringe 512 with connection line 513 positioned in the bottom ofreaction chamber 523. The top phase can be discarded through waste line526. In an alternative embodiment, a centrifuge device is included inreaction device 520 to separate emulsion phases, with a pipetting deviceto withdraw the aqueous phase from the bottom of the centrifuge tube.

To create an emulsion, the appropriate emulsion oil is selected byspatial librarian reagent rail 501 and added to the reaction chamber 523with a vigorous back and forth motion of the solution in the reactionchamber 523.

A moveable magnet device 524 can position a magnet near reaction chamber523 to collect paramagnetic beads to the surface of the reaction chamber523 or retract a magnet to release paramagnetic beads as required. Amagnetic separation (He J. et. al. J Pharm Biomed Anal. 2014;101:84-101.) can be used to change the buffer in a reaction, to removeresidual traces of emulsion oil or breaking solution, to remove anenzyme, to concentrate a product, or to capture nucleic acid or othercomponents onto beads for ease of handling. The surface chemistries ofthe paramagnetic beads and conditions to precipitate, wash, and elutenucleic acids and other biomolecules onto surfaces is well understood,(Boom, W. R. et. al. U.S. Pat. No. 5,234,809. Aug. 10, 1993.), (Reeve,M. and P. Robinson. U.S. Pat. No. 5,665,554. Sep. 9, 1997.), (Hawkins,T. U.S. Pat. No. 5,898,071. Apr. 27, 1999.), (McKernan, K. et. al. U.S.Pat. No. 6,534,262. Mar. 18, 2003.), (Han, Z. U.S. Pat. No. 8,536,322.Sep. 17, 2013.), (Dressman et al., “Transforming single DNA moleculesinto fluorescent magnetic particles for detection and enumeration ofgenetic variation” Proc. Natl. Acad. Sci. 100(15):8817-8822 (2003)),(Ghadessy et al., “Directed evolution of polymerase function bycompartmentalized self-replication”, Proc. Natl. Acad. Sci.98(8):4552-4557 (2000)), (Tawfik and Griffiths, “Man-made cell-likecompartments for molecular evolution” Nat. Biotech. 16(7):652-656(1998)), (Williams et al., “Amplification of complex gene libraries byemulsion FOR” Nat. Meth. 3(7):545-550 (2006)), and many chemistries arepossible and within the scope of the instant disclosure for spatialanalysis. When the nucleic acids and other biomolecules are covalent orotherwise attached to a paramagnetic bead, the beads and attachedbiomolecules can be separated from solution without the additive of aprecipitation solution such as a crowding agent, e.g., polyethyleneglycol, ethanol, etc. If the biomolecules are not attached toparamagnetic beads, reagent rail 501 can add the appropriateprecipitation solution(s) to reaction chamber 523 to force thebiomolecule of interest onto the surface of the paramagnetic bead.

A magnetic separation can be performed using moveable magnet device 524to position a magnet near reaction chamber 523 to collect theparamagnetic beads to a surface of reaction chamber 523 and the reactionmix pumped to waste line 526. A wash solution, such as 80% ethanol, isadded by reagent rail 501 and the beads released by retracting themagnet. In some embodiments, the wash solution can include Triton X-100(Octylphenol ethylene oxide condensate). In some embodiments, the washsolution can include about 0.01% to about 5% Triton X-100. In someembodiments, the wash solution can include about 0.05% to about 1%Triton-X. In some embodiments, the wash solution can include Tris-HCl orTris-EDTA. The beads can be agitated by pumping, magnetic stirrers,bubbling air, or other methods. The beads are recaptured by usingmoveable magnet device 524 to position a magnet near reaction chamber523 to collect the paramagnetic beads to a surface of the reactionchamber 523 and the wash solution pumped to waste line 526. This can berepeated with the same or different wash solutions as needed to purifythe reaction products, to remove unincorporated reactants, to removeemulsion oil or breaking solution, or other reasons. The biomolecule ofinterest can be associated with the bead or with the solution phase asdesired. In addition, reaction chamber 523 can be heated to activate orinactivate enzymes. As is obvious to one skilled in the art, otherdevices and functionality can be added to reaction device 520 includinga centrifuge device or a pipetting device.

In some embodiments, the nanodroplet or bolus generation was performedwith a reversible gelling agent such as ultra low gelling agarose astaught by Geng T. et. al. Anal Chem. 2014; 86(1):703-12. In a preferredworkflow, low melting agarose is added to the microsample before nozzle429 and nanodroplets are produced with at times a single cell and asingle barcoded bead with the cells compartmentalized within nanoliteragarose droplets. The nanoliter agarose droplets are transformed intomicrogels by cooling. The agarose microgels allow small molecules todiffuse into and out of the nanodroplets but retains macromolecules suchas DNA. This enables lysis and removal of PCR inhibitors and PCRamplification (as taught by Geng T. and R. A. Mathies Forensic Sci IntGenet. 2015 January; 14:203-9).

The utility of the nanoliter agarose droplets can be further extended inthe instant disclosure to couple multiple reactions in a row. As needed,the microgels can be harvested and reagents infused. The substrates andenzymes can be changed between reactions, such as a DNA libraryconstruction; see “Library Preparation for Double-Stranded DNA example.”In one example, spatial analysis of polyadenylated mRNA from singlecells and tissue can be performed in nanoliter agarose droplets bylysing cells, capturing mRNA, reverse transcriptase, followed by secondstrand synthesis. In another embodiment, precipitation agents arediffused into the nanoliter agarose droplets to force precipitation forseparations onto the bead inside the nanoliter agarose droplets if thebiomolecule of interest is not attached to the bead. Unbound materialscan diffuse out.

c. Spatial Encoding by Adding Internal Markers or Standards Directly tothe Specimen

In another preferred embodiment, markers, comprised of internal markers,standards, nucleic acids, enzymes, chemicals such as isotopes, masstags, or ratios or mixtures of enzymes, fluorescent markers, Ramanmarkers, optical markers, carbohydrates, lipids, other biomolecules, orchemicals, are added to the specimen in known order by dispensers 335 onthe multifunctional head 330 or alternatively by direct transfer fromprefabricated arrays 900 of markers 901 on a support carrier, backingsurface or membrane (see FIG. 17) attached to and manuevered by themultifunctional head 330. The markers are later analyzed to decode thespatial position in the specimen of the microsample 125. In someembodiments, the decoding can be an orthogonal method to the analyticmethod for the analyte of interest, i.e., a DNA marker could be used todecode spatial information while the sample analysis might be by massspectrometry. In some embodiments, the markers are attached toantibodies or other bioaffinity agents such as to exterior cell surfaceepitopes. In other embodiments, the markers are attached to motifs,compounds, or structures that are transported, electroporated, orotherwise enter into the cells. The markers can be used to identify thespatial position of the microsample 125 and cell for many differenttypes of analyses comprising metabolic characterization and profiling,proteomics, genomics, gene expression, carbohydrate characterization andprofiling, lipid characterization and profiling, and combinations ofanalyses.

Referring to FIG. 17, the markers depicted by letters A to P are eachunique and arranged in patches 901 to form a two dimensional array 900.These markers can be physisorbed as mono- or multilayers onto thesurface of a membrane or support material which is connected to themultifunctional head 330. Alternatively, the markers can be arrayed inform of beads or contained in micro/nanowell architectures, or immersedin hydrogels or other surfaces 660 or structures. During sampling thearray is brought into direct contact with the specimen 301 resulting inthe transfer of marker reagents onto the topmost layer of the specimenthereby encoding the spatial order information directly onto the cells.It is understood that the patch-to-cell registry shown in FIG. 17 is anidealized version. It is more likely that in certain cases multipleadjacent markers will be transferred to the same cell or one marker willbe transferred to multiple adjacent cells which can be used to furtherdefine boundaries between patches.

For spatial analysis of proteins, a dispenser(s) 335 or array 900 on themultifunctional head 330 can apply different solutions of markers toeach microsample 125 on the specimen. For example, mass isotopes orratios of mass isotopes can be added, and, after microsample 125 isprocessed in the Spatial Sampler 300 to produce single cells, theisotopes can be determined by mass spectrometry to determine where inthe sample the cell or cells originated. In other embodiments, themarkers are added as barcodes in the Spatial Encoder module 400.

For nucleic acid analysis, nucleic acids or other markers can be addedto the specimen in known order by dispensers 335 or an array 900 on themultifunctional head 330. The nucleic acids can be electroporated intothe specimen by addition of suitable electrodes and buffer, applied tothe surface, or designed to be uptaken by the cells. Nucleic acidsequences can be added that are not found in the organisms being studiedand their signature used to decode the spatial position of microsample125. In some embodiments, an affinity label, such as biotin, can beattached to the nucleic acid marker and the mating affinity label, suchas streptavidin, attached to beads 630 to capture the nucleic acidmarker after lysis.

The following details one embodiment to add spatial barcodes to thespecimen 301 directly on sampling in the Spatial Sampler module 300.

One embodiment uses a physical array of paramagnetic beads that arepre-arrayed on a Collection multifunctional head 330 with positionalinformation encoded in the primers attached to the beads witholigonucleotides with spatial barcodes 680. This approach greatlysimplifies engineering approaches for the Single Cell Spatial AnalysisSystem 100. The Spatial Sampler module 300 uses the two-dimensionalstage 320 with a multifunctional head 330. Fluidic dispensers 335 firstapplies solution(s) to tissue to dissolve intercellular adhesion.

In this embodiment, the transfer membrane 336 is replaced with acollection head that has an array 900 of paramagnetic beads 630 eachwith a single spatial barcode and in a known order. Referring to FIG.18, the beads can be attached by cleavable linker 620 to backing surface621, alternatively held by magnets located behind backing surface 621 orarrayed and attached by other methods. In one embodiment, each bead canhave two types of biologicals attached: antibodies to binding cellsurface markers 651, 652, and 653 which can all be the same or differentand each individual bead has a DNA oligonucleotide with a unique spatialbarcode 640, 641, and 642, and optional barcodes for cellular identityand molecular identity, and other elements comprised of sequencingprimer, poly T sequence when capturing polyadenylated mRNA, targetedcapture sequence, or other elements. Each bead has a different spatialbarcode marker and is placed in a known order upon backing surface 621.Decoding the spatial barcode in turn allows the sequenced DNA or RNA tobe traced back to its original location in the specimen.

In this embodiment, to collect cells, after dissolution of theextracellular matrix and intercellular adhesion, the collection headwith backing surface 621 of the multifunctional head 330 is pressedagainst the top layer of the tissue and cells bound to the beads byantibodies 651, 652, and 653. To dispense, the multifunctional head 330is moved to the cell input port of the Spatial Encoder module 300 andthe linkers cleaved (or the magnet removed) to deposit all of the beadsinto the input port simultaneously or in groups, with gas pressureapplied if needed. Individual portions do not need to be dispensedbecause the spatial barcode on the beads, which are attached to thecells through the antibody, can be used to decode the position of themicrosample 125 after amplification. This embodiment obviates the needfor spatial barcode reagent rail 401 to add spatially barcoded beads tothe microsamples 125. However, this approach requires a more complicatedmanufacturing process to generate the arrays of spatially encoded beads.

d. Spatial Data Analysis, Representation and Information Description

The Single Cell Spatial Analysis System 100 encodes the physicallocation of microsamples 125 within the specimen 301 to producespatially encoded single cells 1000. Following analysis method 1100,such as DNA sequencing, mass spectrometry, Raman spectroscopy, or otheranalytical methods, single cell analytical data 1600 is produced ofanalytes of interest, such as DNA sequence of target sequences, and atthe same time the encoded spatial information from each sequence can bedecoded to produce decoded single cell spatial information 1500 whichidentifies the spatial position 130 where microsample 125 originated inspecimen 301. The decoded single cell spatial information 1500 can be,for example, 5 nucleotides or ratios of isotopes, or other decoding ofmarkers.

The single cell analytical data 1600 is analyzed by single cell spatialanalysis 2000 software to produce single cell spatial information 3000which has the analytical data and associated physical positioninformation of the microsample 125. For single cell spatial analysis2000, scripts can sort reads by the spatial barcode 607 and reconstruct,for example, the DNA sequence or expression patterns from individualcells from the specimen by position in three dimensions. Types of cells,activity, gene expression, mutation, networks of genes, and expressionpatterns can be mapped to two or three dimensional spatial coordinates.Information from optical or other measured properties can be combinedand represented visually with two and three dimensional plots of theorigin of the microsample and the activity measured, genomic, proteomic,metabolomics, systems biology, etc.

The data analysis can build upon existing analytic platforms. Forexample, downstream (N)NGS workflow can use existing (N)NGS sequencinganalysis and bioinformatics. For the bioinformatics pipeline, thequality of the data is assessed and, optionally, the reads trimmed andthose with poor quality filtered out. High quality reads are aligned tothe reference genome or transcriptome using one of the many availablehigh-throughput sequencing mapping tools. Alignments are assembled intofull-length transcripts or contigs based on a reference genome. For geneexpression, the aligned reads are subsequently passed to quantificationtools to obtain a measure of expression. After the completion of thesemain steps, several differential analyses can be executed to identifydifferentially expressed genes and transcripts. For all reads, thespatial barcode 607 information in either the same analysis or anorthogonal analysis are used to decode the origin of the same andprovide input into the creation of single cell spatial information 3000,which combines single cell analytical data with the spatial data.

e. Examples of Workflows and Applications

Many applications are enabled by the Single Cell Spatial Analysis System100. The capability of analyzing the identity, genetic sequence, geneexpression, proteomic and metabolomic profiles of specimens and howgroups of cells function in three dimensional matrices can be applied toforensics, molecular diagnostics, pathology, cell biology, celldiscovery, histology and many other applied areas to understanding ofhow tissue functions. Spatial analysis of tissue at the single celllevel will provide extraordinary insight and understanding, tissuecomposition of different cell types, structure, and how cells functionin a microenvironment. Animal, plant, and microbial communities, such asbiomes and films, will be important applications and how biomes changeat interfaces of different organs and microenvironments. The Single CellSpatial Analysis System 100 can be applied to molecular diagnosticswhere spatial analysis of biopsies can provide information about thestate of the tissue, subregions 150 of activity, such as cancerousgrowths, and sources of qualified material for treatment; many raw andprocessed samples can be addressed including blood, forensic samples,tissue samples, fine needle aspirates and many others. In many ofexamples, the production of single cells with spatial barcodes isdiscussed. It will be clear to one skilled in the art that ensemblemeasurements of groups of cells with the same spatial barcodes, such asfor a microsample 125, is within the scope of the present disclosure.

Examples are discussed in the following sections. Many otherpermutations, workflows, modules, devices, and combinations arepossible. The names of the modules and subsystems are not limiting andfunctionality can be distributed differently between modules.

i. Example: Spatial Analysis of Polyadenylated mRNA from Single Cellsand Tissue.

mRNA analysis from single cells has been developed and workingconditions based upon the pioneering works of Macosko E. Z. et. al.Cell. 2015; 161(5):1202-14 and Klein A. M. et. al. Cell. 2015;161(5):1187-201 and others cited therein are incorporated by reference,including instrumentation, chemistry, workflows, reactions conditions,flowcell design, and other teachings. However, no information aboutwhere in the specimen the cells originated is encoded with currentmethods and therefore all spatial information about the cell's physicalposition in the specimen and the identity of cells located near eachcell are lost. The instant disclosure encodes the spatial position 130to allow increased understanding of the identity of each cell, its stateof gene expression, and the network of interactions in specimen 301 tobe better understood.

A preferred workflow for mRNA to encode spatial position of themicrosample 125 into DNA is shown in FIG. 19. The workflow begins withsampling from tissue or other specimens 301 to collect microsamples 125,i.e., cells and groups of cells, in known physical order, one layer at atime; the layer can be a single layer of cells or multiple layers ofcells. In a preferred embodiment, the microsample 125 is then input intoa fluidic flow system in known order in the Spatial Sampler module 300.After the spatially encoded bead(s) has been added in the SpatialEncoder module 400, the microsample 125 and beads pass through nozzle429 which then generates nanodroplets or small boluses each withpreferably a single bead and one or less cells. In the Spatial LibrarianSubsystem 500 the cells are lyzed, and the released mRNA captured ontothe barcoded oligonucleotide on the bead 680. A reverse transcriptionreaction is performed either in the bolus or after pooling nanodropletsor boluses. The reverse transcription reaction uses the oligonucleotideas a primer and synthesizes complementary DNA (cDNA) to the mRNAsequence attached to one strand of the spatial encoded oligonucleotide,thereby covalently linking the cDNA to the DNA of the oligonucleotide.Since the oligonucleotide contains a spatial barcode, the cDNA now has aspatial barcode which can be read out by DNA sequencing after librarypreparation.

In other embodiments, the microsample 125 can be output into a well suchas a microtiter plate or test tube, or into a flow cell, or otherordered substrate in known order from the Spatial Sampler module 300. Ina preferred embodiment, a spatially encoded bead or set of beads allwith the same spatial barcode is added to the microsample 125 or tosingle cells or groups of cells from the microsample 125. Alternatively,the spatial encoding can be on nucleic acid attached to the surface of aflowcell, or on a hydrogel, or on the interior of a capillary ormicrochannel, or other surfaces or matrices; these alternatives are allwithin the scope of the present disclosure when the term bead is used.

A preferred embodiment for spatial analysis of mRNA is described in moredetail. In one embodiment, single channel fluidics are used. Referringto FIG. 20, a bolus of 100 nL of spatially barcodedoligonucleotide-functionalized beads 680 with a poly T sequence as thecapture region 610 with a single spatial barcode is added in junction414 to 100 nL of microsample 125 from spatial sampler outputmicrochannel 386 and lysis/reverse transcriptase mix added separatelythrough spatial encoder reagent syringe pump 418 and reagent connectingmicrochannel 419; the lysis conditions and reverse transcriptasedescribed by (Fekete R. A. and A. Nguyen. U.S. Pat. No. 8,288,106. Oct.16, 2012) are incorporated by reference. Monodisperse nanodroplets fromsingle cells with spatially coded beads with lysis/RT mix, are producedusing a nozzle 429 (Macosko E. Z. et. al. Cell. 2015; 161(5):1202-14.)(Klein A. M. et. al. Cell. 2015; 161(5):1187-201.) (Geng T. et. al. AnalChem. 2014; 86(1):703-12) and output through spatial encoder outputmicrochannel 430.

After processing with the Spatial Preparation Subsystem with spatiallybarcoded oligonucleotide-functionalized beads 680 with a poly T sequenceat capture region 610, the nanodroplets or boluses with a distributionof beads and single cells can be input in the Spatial LibrarianSubsystem 500. Cells are lysed by heating or other methods andpolyadenylated mRNA 681 captured onto the oligonucleotide to formcaptured mRNA structure 682. The oligonucleotides 601 are barcoded forspatial barcode information 607 as well as cellular barcodes 608 andmolecule barcodes 609. Amplification primer 604 and sequencing primer605 may be included on the oligonucleotide, or may be added indownstream library preparation methods as needed. The amplificationprimers can be for T7 polymerase for amplified RNA production (VanGelder R. N. et. al. Proc Natl Acad Sci USA. 1990; 87(5):1663-7.), PCR,rolling circle transcription-based amplification, rapid amplification ofcDNA ends, continuous flow amplification, and other amplificationmethods.

After lysis and capture of the mRNA onto the poly T, a reversetranscriptase reaction is performed in Library Preparation Module 500 toproduce cDNA attached to bead 683, formed from the mRNA, and nowcontaining spatial, cellular, and molecular barcodes in addition to anysequencing and amplification primers and is attached to the bead 680through a cleavable linker. Cleavage of the linker can release the cDNAfrom the bead when desired. A photocleavable or chemical cleavablelinker and fluorescent tag(s) to aid in quality control and processdevelopment is included in the instant disclosure. As required,fragmentation of the RNA or cDNA can be performed using methodscomprised of chemical, biochemical, and physical methods.

The reverse transcription reactions can be assayed by qPCR usingreagents added from spatial librarian reagent rail 501 to reactionchamber 523 with thermal cycling from temperature control device 521with optic device 522 monitoring the qPCR reaction. cDNA produced bycleaving the fluorescently tagged cDNA can be analyzed by QC device 525with capillary electrophoresis. External polyadenylated transcripts250-2,000 nt in length at a 10⁶ range of concentrations can be addedfrom spatial librarian reagent rail 501 to assess the dynamic range andrange of detection with NGS analysis (ERCC RNA Spike in Controls,4456740, Life Technology).

Alternative preferred embodiments include performing an RNA ligasereaction to covalently join the mRNA to one strand of the doublestranded oligonucleotide after lysis and capture of the mRNA ontospatially barcoded oligonucleotide-functionalized beads 680 with a polyT sequence as the capture region 610, or ligating RNA to a singlestranded RNA or DNA attached to the bead.

In some embodiments, the emulsion is broken at this stage in ademulsification step. The emulsion can be broken, for example by heatingthe mixture, acidification, centrifugation and ultrasonic treatment.When nanoliter agarose droplets are used, the solidified nanoliteragarose droplets can be separated from the oil with a filter, e.g., a 40μm or other pore size filter, incorporated into reaction chamber 523 andwashed with water or solvents from spatial librarian reagent rail 501.The agarose can be melted when required to release the bead with spatialinformation encoded. When nanoliter aqueous droplets or boluses areused, the demulsification can be performed in the reaction chamber 523or in some embodiments externally using chemistry and process flows asknown to the skilled artisan (Kasinskas, R. et. al. WO 2012138926 A1)(Xu, M. et. al. J. Gen. Eng. Biotech. 10, 239-245, 2012) includingadding solvents, aqueous solutions, non-aqueous solutions, boluses ofaqueous solutions interspersed with non-aqueous solutions, while usingmagnetic separations, filters, membranes, charge, or other properties,to retain the spatially encoded beads while the emulsification agentsare removed. Multiple solutions can be added in appropriate order.

In some embodiments, nanoliter droplets or boluses are broken and thennew nanodroplets or boluses are created for a second time preferablywith a single bead in each nanoliter droplet or bolus in anemulsification agent.

Following cDNA synthesis, the reverse transcriptase can be heatinactivated and/or depending on the downstream chemistries, the cDNAbound to the bead can be purified by magnetic separation, whenparamagnetic beads are used, to remove inactive reverse transcriptaseand change buffers and purify double stranded cDNA attached to bead 684.

Second strand synthesis of DNA can be performed by adding second strandsynthesis reaction mix and DNA polymerase or enzyme mix in appropriatevolumes to reaction chamber 523 from spatial librarian reagent rail 501and incubating the reaction to produce double stranded cDNA attached tobead 684. For example, NEBNext Second Strand Synthesis Reaction Bufferand NEBNext Second Strand Synthesis Enzyme Mix NEB #E6111S may be usedwith incubation at 16° C. for 2.5 hr. A magnetic separation can be usedto purify the double stranded DNA and remove reactants and enzymes.Double stranded cDNA attached to bead 684 can be processed in downstreamlibrary preparation for double stranded DNA, as per the section ‘LibraryPreparation for Double-Stranded DNA example’ and other methods ofpreparing NGS and (N)NGS sequencing libraries either in the SpatialLibrarian subsystem 500 or externally.

The cleavable linker can be cleaved if desired to produce doublestranded cDNA released from the bead 685 from double stranded cDNAattached to bead 684 or to produce cDNA attached to bead 682. Thedouble-stranded DNA released from the bead 685 is ready for qualitycontrol and may include a fluorescent or other tag.

ii. Library Preparation for Double-Stranded DNA Example.

Double-stranded DNA can be prepared into a library ready foramplification and (N)NGS sequencing. The double stranded DNA can beattached to a paramagnetic bead through an oligonucleotide or doublestranded DNA in solution can be used with addition of paramagneticbeads.

One embodiment of the workflow is illustrated in FIG. 21.Double-stranded DNA attached to a bead, such as double stranded cDNAattached to bead 684, can be end-polished in reaction chamber 523 byaddition of reaction mix and enzymes, for example, the NEBNext® EndRepair Module (NEB E 6050S) reagents, from reaction rail 501 to generateend-polished DNA product 810, an end-polished, blunt-endeddouble-stranded DNA having 5′-phosphates and 3′-hydroxyls; other kitssuch as Agilent PCR polishing kit 200409 and other enzymology canperform the same function. Following end polishing, a magneticseparation is performed in reaction chamber 523 to remove reactants andenzymes from end-polished DNA product 810.

Following polishing, A-tailing is used to generate fragments ready toligate with a primer with a complementary T overhang and to preventconcatamer formation during ligation. A-tailing can be performed usingcommercially available kits such as the NEBNext® dA-Tailing Module (NEBE6053S) with enzyme and master mix added from the spatial librarianreagent rail 501 to reaction chamber 523 containing end-polished DNAproduct 810 and incubating the reaction to produce blunt-endeddouble-stranded DNA having 5′-phosphates with an A residue overhang onthe 3′ end, A-tailing DNA product 815. Following A tailing, a magneticseparation is performed in reaction chamber 523 to remove reactants andenzymes from A-tailing DNA product 815.

A double stranded second primer 611 with a complementary T overhang canbe ligated by DNA ligase onto the 3′ end of A-tailing DNA product 815which in some embodiments is attached to a bead or surface 660. DNAligase, DNA ligase reaction mix, and second primer 611 are added fromspatial librarian reagent rail 501 to reaction chamber 523 andincubating the reaction. DNA ligation can be performed usingcommercially available kits or reactions, e.g. NEBNext® Quick LigationModule, NEB E6056S. Following DNA ligation, a magnetic separation isperformed in reaction chamber 523 to remove reactants and enzymes. Theproduct is now a double stranded DNA product 820 that has incorporatedon one end the oligonucleotide 601, which can still be attached to bead630, and the other end has incorporated second sequencing primer 611.Oligonucleotide 601 can have a cleavable linker 602, attached to anamplification primer 604 with fluorescent label 603, a sequencing primer605, barcode region 606, and capture region 610 and other functionalitysuch as affinity tags, e.g., biotin and others as described in Uhlén, M.BioTechniques. 2008. 44:649-654. The barcode region is comprised of aspatial barcode 607, cellular barcode 608, and molecular barcode 609.

Second primer 611 can contain functionality comprising a secondsequencing primer 612, second barcode region 613, second amplificationprimer 614, and additionality functionality such as affinity tags.

In one embodiment of the present instant disclosure, the double strandedDNA product 820 is attached to beads and then released by cleavage ofcleavable linker 602 and eluted with water, buffer, or other liquidswhile magnets hold the paramagnetic beads to produce double stranded DNAproduct in solution 830. This further purifies the double stranded DNAproduct in solution 830 which can be output for analysis with NGS, NNGS,nanopore, electrochemical, Sanger sequencing, single moleculesequencing, or other genetic analysis systems. If a fluorescent label603 is attached, the cleaved product can be analyzed either on thedevice using quality control device 525 or collected and analyzed off ofthe Single Cell Spatial Analysis System 100. The analysis results can beused to assess the quality and quantity of double stranded DNA product830.

In one embodiment of the present instant disclosure, second primer 611is attached to a bead attached to second primer 650 which may havesimilar or different properties such as different magnetic moment thanbead 630. For example, bead attached to second primer 650 may have adifferent size, magnetic moment, surface coating, affinity tags, orother property(s) that allows bead attached to second primer 650 to bemanipulated differently than bead or surface 630. The difference inproperties can be manipulated to purify double stranded DNA product 820which is attached to bead 650 through the second primer.

In another embodiment of the present instant disclosure, second primer611 is attached to a surface 660. The surface can be a flow cell 670. Inone embodiment, the product of the A-tailing reaction, the blunt-endeddouble-stranded DNA having 5′-phosphates and an A residue overhang onthe 3′ end, A-tailing DNA product 815, is cleaved off of the bead 630 toproduce freed A-tailed DNA product 816 and introduced to a surface 660or flow cell 670 which has second primer 611 with a T overhang bound tothe surface 660 or to flow cell 670. DNA ligase and DNA ligase reactionmix are added to produce a double stranded DNA product 840 that hasincorporated on one end the oligonucleotide 601 except the cleavablelinker and the other end has incorporated sequencing primer 611 attachedto surface 660 or flow cell 670. Oligonucleotide 601 can be comprised ofamplification primer 604, fluorescent label 603, a sequencing primer605, barcode region 606, and capture region 610 and other functionalitysuch as affinity tags. The barcode region is comprised of a spatialbarcode 607, cellular barcode 608, and molecular barcode 609.

iii. Example: Spatial Analysis for Whole and Partial Genome Sequencingfrom Single Cells in a Specimen.

The Single Cell Spatial Analysis System can spatially encode cellularlocation in specimen 301 for whole genome DNA sequencing applications byaddition of spatial barcodes 607. As described in this instantdisclosure, single cells and groups of cells from subregions 150 of aspecimen 301 can be input as an individual microsample 125; the analysiscan be using whole or partial genome sequencing.

In one preferred embodiment, referring to FIG. 22, the Spatial Samplermodule 200 collects microsamples 125 from specimen 301 as described, orother embodiments. The Spatial Encoder module 400 adds beads witholigonucleotides with spatial barcodes 680 or other surfaces withspatial barcoding in known order as nanodroplets or boluses are producedin Spatial Encoder module 400. In other embodiments, spatial barcodesare added directly to the specimen as described in the “Spatial EncodingBy Adding Internal Markers or Standards Directly to the SpecimenDescription”.

The Spatial Library module 500 performs the chemistry on thenanodroplets or boluses from the microsamples 125. For example for DNAsequencing, the microdrops can be formed with cell lysis buffer such as0.5% sodium dodecyl sulfate (SDS), 0.1 mg/mL proteinase K, 100 mM EDTA,and 10 mM Tris-HCl. After lysis, if low melting point agarose is used,the temperature can be decreased to gel the agarose and the gel dropletscan be repeated washed. A restriction digest can then be performed bymixing a restriction digest buffer, for example, 100 mM potassiumacetate, 25 mM Tris-acetate, pH 7.6, 10 mM magnesium acetate, 10 μg/mlBSA, 0.5 mM β-mercaptoethanol, containing a blunt end restriction enzymeor a restriction enzyme that generates an overhang with the beads. Afterdiffusion, emulsion oil can be added and the temperature can be raisedto 37° C.; the digestion of the genomic DNA will occur to produce bluntended fragments or fragments with overhangs.

In one embodiment, the oligonucleotide has a cleavable linker 602,attached to an amplification primer 604 with fluorescent label 603, asequencing primer 605, barcode region 606, and capture region 610. Inone embodiment, the capture region 610 is blunted ended while in otherembodiments the capture region 610 is single-stranded to capturespecific or non-specific DNA sequences. The barcode region is comprisedof a spatial barcode 607, cellular barcode 608, and molecular barcode609. In some embodiments, the molecular barcode 609 is not used. Whenrestriction enzymes are used, the oligonucleotide on the bead can bedesigned to not contain the relevant restriction site.

When the agarose encapsulation is used, after restriction digest, thetemperature can be lowered to gel the agarose, and after an optionalrinse step(s), either DNA polymerase with appropriate reaction mixtureor DNA ligase with appropriate reaction mixture is added. Afterdiffusion of the DNA polymerase or ligase and mixture into the gelledmicrodroplets, emulsion oil is added and the temperature raised, forexample to 37 C. DNA captured using overhangs or hydridization isreplicated with a DNA polymerase to incorporate the spatial barcode 607into the DNA strand in the microdroplet. For blunt ended capture, DNAligase incorporates a spatial barcode 607 on a blunted ended bead withspatial barcode 680 with the ligated DNA strand in the nanodroplet,bolus, or well. The microdroplets or microsamples 125 in wells can bepooled for the remaining library preparation. For single strandedcapture regions, the length of the capture region and hybridizationconditions can be adjusted to tune the specificity of capture. In somecases, related sequences might be desired to be captured while in othersincreased specificity of capture may be desired: either can beaccommodated including a plurality of capture sequences comprised ofsequences that interrogate different signatures, networks, diseasesincluding cancer, microbes, genetic traits, introns, exons, and groupsof sequences without limitation.

DNA or RNA can be fragmented in the Spatial Library module by methodswell known to one skilled in the art, using chemical, biochemical, orphysical fragmentation when required. In one embodiment, the SpatialLibrarian Subsystem 500 adds restriction enzymes to double strandedwhole genome DNA to create restriction fragments with specificsequences. The restriction fragments can have an overhang that can becaptured or ligated to its complementary sequence on the capture region610 on the oligonucleotide. In another embodiment, the Spatial LibrarianSubsystem 500 adds polishing reagents to fragments DNA to produce bluntended fragments.

iv. Example: Spatial Encoding Nucleic Acids with Targeted Sequencing

The Single Cell Spatial Analysis System 100 can spatially encode wherein the specimen 301 the cell was located to prepare libraries fortargeted DNA sequencing. The overall workflow for one embodiment isshown in FIG. 23. Beads with oligonucleotides with spatial barcodes 680or other surfaces 660 with spatial barcoding are used with captureregion 610 designed to be complementary to the DNA sequence to becaptured. In another example, an overhang such as a restriction site canbe captured or ligated to its complementary sequence. The sequences tobe captured can be generated by using a restriction digest mixture toform the microdroplets and then performing the digest in themicrodroplets and hybridization. In another embodiment, themicrodroplets are formed with fragmentase such as NEBNext® dsDNAFragmentase and the DNA is fragmented. The length of the capture regionand hybridization conditions can be adjusted to tune the specificity ofcapture. In some cases, related sequences might be desired to becaptured while in others increased specificity of capture may bedesired: either can be accommodated. A plurality of capture sequencescomprised of sequences that interrogate different signatures, networks,diseases including cancer, microbes, genetic traits, introns, exons, andgroups of sequences without limitation are within the instantdisclosure.

The targeted DNA captured is replicated with a DNA polymerase, e.g.,phi29, Taq, or others, to incorporate spatial barcode 607 into thecaptured targeted DNA strand in the nanodroplet, bolus, or well. Thecaptured DNA with the spatial barcode 607 on beads can be pooled andprocessed into (N)NGS libraries either in the Single Cell SpatialAnalysis System or externally, with amplification when required,comprised of PCR, rolling circle amplification (RCA), Loop mediatedisothermal amplification (LAMP), Helicase-dependent amplification (HDA),Nicking Enzyme Amplification Reaction (NEAR) and other methods.

v. Example: Library Preparation Using Transposons.

Libraries for (N)NGS can be prepared using tagmentation with transposonsincluding the Nextera Tagmentation(http://www.epibio.com/docs/default-source/protocols/nextera-dna-sample-prep-kit-(illumina—compatible).pdf?sfvrsn=4).In this embodiment, referring to FIG. 24, double stranded DNA withspatial encoding attached to a bead or surface is used as the input inthe Spatial Library Subsystem 500. Once the double stranded DNA isproduced in reaction chamber 523, transposons, e.g., Nextera enzyme,reaction mix, and water are added from reagent rail 501. The reaction isincubated for example at 55 C for 5 min. A bead purification isperformed to remove reactants and purify the double stranded productwith transposon inserted 840. In the example shown in FIG. 24,precipitation agents are not added since the desired material isattached to a bead or surface; the inclusion or not of precipitationreagents is not limiting. Reagent rail 501 is used to add Nuclease-FreeWater, Nextera Adaptor 2 (or other barcoded adapters), Nextera PCREnzyme, PCR Buffer, Nextera Primer Cocktail. Nine cycles of PCR can beperformed. A bead purification is performed to remove reactants andpurify the double stranded DNA product 850 before elution into buffer orwater. The double stranded DNA product 850 library is now ready to QCand bridge amplification on the flow cell. Many variations of the methoddescribed here are within the instant disclosure and are obvious to oneskilled in the art.

In another embodiment, the double stranded DNA with a spatial barcode isattached to a flow cell 670 or surface 660. In this example, samechemistry as shown in FIG. 24 could be applied except the products areattached to the flow cell 670 and flow cell 670 is washed rather thanthe beads.

vi. Example: Single Cell Spatial Systems Biology

Single cell spatial analysis 1000 and its integration into a Single CellSpatial Analysis System 100 can be applied to systems biology. Systemsbiology connotes the integration of two or more data streams fromspecimen 301, e.g., DNA sequencing with RNA sequencing; DNA and RNAsequencing with mass spectrometry for proteins, DNA sequencing withmetabolomics; and other combinations without limitation. In the instantdisclosure, the benefits of single cell spatial analysis 2000 includeall the benefits of systems biology extended with the benefits ofunderstanding the identity, location, activity, and interplay of singlecells in a matrix such as tissue, organs, organisms, biofilms, moleculardiagnostics samples, and in the environment.

1. Spatial Encoding DNA and RNA from Single Cells Simultaneously

It is frequently of interest to sequence both the DNA and RNA fromsingle cells and in the instant disclosure with information of thespatial position 130 of microsample 125 in specimen 301. In thisworkflow, the paramagnetic beads can be a mixture of, for example, twotypes of beads with oligonucleotides with spatial barcodes 680 with acapture region 610 with one type of bead having poly T sequences and theother type sequences for targeted DNA capture. The beads can besynthesized by split pool, and mixed in the appropriate ratios.

In the workflow to spatially encode both DNA and RNA, the cells arelysed and the DNA and RNA bound by hybridization. Reverse transcriptionis performed to convert the bound mRNA into cDNA. Second strandsynthesis can convert the cDNA to double stranded DNA and at the sametime incorporate the target DNA captured to the oligonucleotide attachedto the bead. The beads can then be pooled, the emulsion broken, andlibraries created. On sequencing, the molecular barcodes and string ofTs or As will identify RNA products for quantitation of gene expression.

2. Simultaneous Spatial Analysis of Nucleic Acids and Other Analytes.

An alternative configuration of single cell spatial analysis 2000 is toanalyze nucleic acids simultanteously with other cellular componentscomprised of proteins, lipid, carbohydrates, metabolites,etc. Thespatial encoding can be any method including DNA analysis or by addinginternal markers or standards directly to specimen 301. In a preferredembodiment, markers, e.g., internal markers, standards, nucleic acids,chemicals etc., are added to the specimen in known order by dispensers335 on the multifunctional head 330. In some embodiment single cellsfrom a microsample 125 with spatial barcoding are prepared as describedand nanodroplets with a single cell and single bead with a singlespatial barcode are produced. The sample can be processed by twoanalysis modulaties. For example, when polyadenylated mRNA is analyzed,after cDNA synthesis, if a magnetic separation is performed, thematerial unbound to the paramagnetic bead can be analyzed by aorthogonal method such as an mass spectrometry to determine proteomicsand the double stranded cDNA attached to bead 684 can be used for mRNAanalysis with nucleic acid decoding. One or both sets of markers can beanalyzed to decode the spatial position in the specimen of themicrosample 125.

In some embodiments, the decoding can be an orthogonal method to theanalytic method for the analyte of interest, i.e., a DNA marker could beused to decode spatial information while the sample analysis might be bymass spectrometry. In some embodiments, the markers are attached toantibodies such as to exterior cell surface epitopes. In otherembodiments, the markers are attached to motifs, compounds, orstructures that are transported, electrophorated, or otherwise enterinto cells. The markers can be used to identify the spatial position 130of the microsample 125 for many different types of analyzes comprisingmetabolic characterisation and profiling, proteomics, genomics, geneexpression, carbohydrate characterisation and profiling, lipidcharacterisation and profiling, and combinations of analyzes.

In some embodiments, the spatial encoding of single cells or groups ofcells is only encoded with one modality. For example, single cellspatial analysis 1000 of specimen 301 can determine through nucleic acidsequencing the spatial information needed to identify the microsample125 and individual cells. The nucleic acid sequencing provides thespatial information which can then be used even when the same sample isanalyzed by a different modality, such as mass spectrometry, to providespatial information of where the microsample 125 was in specimen 301since the order of the barcoded microsample 125 can be tracked. In theseembodiments therefore only one barcode is required for systems biologyor other applications.

vii. Example: Forensics Applications

For forensics, specimens 301 can be recovered from crime scenes by tapelifts, swabs, and other collection devices from a diverse set of sampletypes, such as blood, semen, sputum, etc., on a diverse set of surfaceswith possible mixtures of cells from different contributors. Specimen301 can be input into the Single Cell Spatial Analysis System 100 andthe pattern of cells from the two-dimensional or three-dimensionalspecimen analyzed by methods comprised of short tandem repeats (STRs),such as the Combined DNA Index System (CODIS) or other STR panels, forsingle nucleotide polymorphisms (SNPs), gene sequencing, proteinprofiling, and other methods. Specimen 301 can be directly contacted bya portable Single Cell Spatial Analysis System 100 or Spatial SamplingModule 300.

Single cell analysis can identify the contributors to a mixture,regardless of the number of contributors. In addition to identifyingcontributors to mixtures, spatial analysis retains information of thepattern of the cells which may contain important evidence about thecrime and how it was reflected in the crime scene. The patterns revealedby single cell spatial analysis 1000 may show layers, edges of contact,spatter, or other information. SNP analysis with single cell spatialanalysis 1000 will allow facial reconstruction of victim, suspects, andcontributors (Claes P. et. al. PLoS Genet. 2014; 10(3):e1004224.) as thefield continues to advance.

viii. Example: Portable Single Cell Spatial Analysis System

A portable Single Cell Spatial Analysis System 100 will enable directsampling and single cell spatial analysis 1000 at a non-laboratorylocation. In one configuration of the Spatial Preparation Subsystem 200,multifunctional head 330 directly contacts specimen 301 when thespecimen is a surface, material, or other two or three dimension matrix.The multifunctional head 330 enables the application of stain to thesample, for example, to identify regions with human cells at a crimescene, regions with malignant cells, live/dead, or other attributes. Theappropriate subregions can be transferred by transfer membrane 336 intothe Single Cell Spatial Analysis System 100 for preparation of samplesfor single cell spatial analysis 1000.

A portable Single Cell Spatial Analysis System 100 will enable singlecell spatial analysis 1000 while the system is physically moved on itsway to an analysis system. Alternatively the spatial encoded samplemight be produced at the non-laboratory location and transported to ananalysis system in a cartridge 4000. A sample-to-answer portable systemcan be produced by combining the Single Cell Spatial Analysis System 100with a miniaturized detection system such as Oxford nanopore sequencing,a portable mass spectrometer, Raman, capillary electrophoresis,real-time PCR, or other miniaturized detection system.

f. Integration with Upstream and Downstream Analysis

The Single Cell Spatial Analysis System 100 or a portable version can beintegrated with upstream specimen processing and downstream samplepreparation and analysis processing in a preferred embodiment. Forupstream specimen processing, that is processing before transferring anyof specimen 301, the Single Cell Spatial Analysis System 100 in oneembodiment adds reagents to the specimen and optionally measuresattributes with optics head 331 such as fluorescence, colorimetric,Raman, Surface Enhanced Raman, or other optical properties. Thisupstream information can inform whether subregions 150 are of interestin the specimen and/or the workflow to be performed. In anotherembodiment, multifunctional head 330 has an additional sensor such thatcan perform electrophysiology measurements before destructive transferof microregions 150 of specimen 301. In another embodiment, the SingleCell Spatial Analysis System 100 collects raw samples with a device thatproduces specimen 301 from tissue or other matrices, for example,sectioning tissue, contact transferring, or by dissolution to createmicrosamples 125.

In another embodiment, downstream analytical subsystems are incorporatedsuch as a DNA sequencer, mass spectrometer, real time PCR, singlemolecule, capillary array electrophoresis, DNA or protein microarray, orother analytical systems. For nucleic acid analysis, a sample-to-answersystem embodiment can be produced by combining a Single Cell SpatialAnalysis System 100 with the Spatial Librarian subsystem 500 with aminiaturized detection system such as nanopore sequencing, capillaryarray electrophoresis, or real time PCR system.

What is claimed is:
 1. A system comprising: (i) a biological specimen;and (ii) added to each of a plurality of different microsamples from thebiological specimen, a marker comprising spatial information thatencodes the original spatial position of the microsample within thebiological specimen.
 2. The system of claim 1, wherein the biologicalspecimen comprises human tissue, animal tissue, or plant tissue, abiopsy, a cellular conglomerate, an organ fragment, an organism, wholeblood, bone marrow, biome, a biofilm, a fine needle aspirate or anyother solid, semi-solid, gelatinous, or frozen three dimensional or twodimensional matrix of biological nature.
 3. The system of claim 1,wherein the microsamples comprise a single cell or a plurality of cells.4. The system of claim 1, wherein the marker comprises a polynucleotide.5. The system of claim 4, wherein the nucleic acid is bound to amembrane, chip surface, bead, surface, flow cell, or particle or isindirectly bound via an adapter molecule e.g., a complementary nucleicacid or a chemical crosslinker.
 6. The system of claim 1, wherein themarker comprises a peptide, antibody, protein, small molecule, isotopesuch as lanthanide, Raman marker, mass tag, fluorescent orchemiluminescent probe.
 7. The system of claim 1, wherein themicrosamples are dissociated from the biological specimen.
 8. The systemof claim 7, wherein the microsamples are entrained in microdrops in afluidic stream.
 9. The system of claim 7, wherein the microsamples aresupported by at least one substrate, e.g., a membrane.
 10. A device forthe analysis of a biological sample, the device comprising: a samplemodule configured to extract microsamples from a biological specimen;and a recipient module configured to receive the microsample biologicalspecimen from the sample module for analysis.
 11. The device of claim10, wherein the recipient module performs a downstream analysis selectedfrom nucleic acid sequencing, next generation sequencing, next nextgeneration sequencing, proteomic, genomic, gene expression, genemapping, carbohydrate characterization and profiling, lipidcharacterization and profiling, flow cytometry, imaging, microarray,metabolic profiling, functional, or mass spectrometry or combinationsthereof.
 12. A device comprising: an element selected from a membrane,filter, surface, capillary, microchannel, device, and microfabricatedchip; and means to bring the element into direct contact or closeproximity to a biological specimen for the purpose of labeling orextracting a plurality of microsamples in an order based on theiroriginal spatial position within the biological specimen.
 13. A systemcomprising: a stage for supporting a biological specimen; a devicecomprising an array of markers comprised in beads, surfaces, flat ormicrofabricated structures; means for transferring the array of markersinto or onto the biological specimen at predetermined spatial positions.14. A method comprising: adding, to each of a plurality of differentmicrosamples from a biological specimen, a marker comprising spatialinformation that encodes the original spatial position of themicrosample within the biological specimen.
 15. The method of claim 14,further comprising dissociating the microsamples from the biologicalspecimen.
 16. The method of claim 15, comprising adding the markers tothe microsamples before dissociating the microsamples from thebiological specimen.
 17. The method of claim 15, comprising adding themarkers to the microsamples after dissociating the microsamples from thebiological specimen.
 18. The method of claim 15, wherein eachmicrosample comprises a single cell.
 19. The method of claim 15, whereineach microsample comprises a plurality of cells.
 20. The method of claim15, wherein dissociating the microsamples comprises extracting themicrosamples in a raster pattern across the biological specimen.
 21. Themethod of claim 15, wherein the microsamples are dissociated in a 3-Dpattern.
 22. The method of claim 15, wherein dissociating comprisescontacting the biological specimen with a membrane, applying vacuum tothe membrane to hold a layer comprising the microsamples; and removingthe microsamples held by the membrane from the biological specimen. 23.The method of claim 22, comprising removing a second layer of themicrosamples from the biological specimen after a first layer isremoved.
 24. The method of claim 15, further comprising moving thedissociated microsamples into a fluidic stream.
 25. The method of claim24, wherein the microsamples are moved into the fluidic stream in anorder correlated with their original spatial position in the biologicalspecimen.
 26. The method of claim 24, wherein microsamples areincorporated into microdrops (e.g., nanodroplets or boluses) in thefluidic stream.
 27. The method of claim 26, wherein the microdropscontain one or more beads.
 28. The method of claim 27, wherein the beadsare paramagnetic.
 29. The method of claim 27, wherein the beads arefunctionalized with oligonucleotides comprising the spatial informationin the form of a nucleotide barcode.
 30. The method of claim 29, whereinthe nucleotide barcode is unique for each cell or group of cells in themicrosample.
 31. The method of claim 29, wherein the oligonucleotidecomprises barcodes for cellular, molecular, or quality control purposes.32. The method of claim 29, wherein the nucleic acid of the samplecomponent including but not limited to groups of cells or single cellsis enzymatically combined with the oligonucleotide of the bead.
 33. Themethod of claim 29, wherein the nucleic acid is subjected to librarypreparation and nucleic acid sequencing.
 34. The method of claim 29,wherein the oligonucleotide further comprises a poly T tail, and themethod comprises capturing mRNA molecules from the microsamples having apoly T tail; and reverse transcribing the mRNA molecules to produce cDNAmolecules comprising the barcode where the barcode provides the spatialinformation.
 35. The method of claim 29, wherein the oligonucleotidefurther comprises a capture sequence complementary to a target sequence,and the method comprises capturing DNA molecules from the microsamplehaving the target sequence; and extending the oligonucleotide to producea nucleic acid molecule having a copy of the target sequence andcomprising the barcode, wherein the barcode provides the spatialinformation.
 36. The method of claim 29, wherein dissociating comprisescontacting the biological sample with a cell dissociation solutioncomprising at least one protease that digests extracellular matrix. 37.The method of claim 36, wherein the at least one protease is selectedfrom collagenases, elastase, trypsin, papain, hyaluronidase,chymotrypsin, neutral protease, clostripain, caseinase, neutral protease(Dispase®), DNAse, protease XIV.
 38. The method of claim 36, wherein thecell dissociation solution is in the form of a fluid, mist, fog, oraerosol applied to the biological sample.
 39. The method of claim 29,further comprising decoding the spatial information in the microsamplesto determine the original spatial position of each microsamples.
 40. Amethod comprising: providing a biological specimen; collectingmicrosamples from each of a plurality of different spatial positions inthe biological specimen; attaching to nucleic acids in each microsamplea marker comprising a nucleic acid barcode comprising spatialinformation that encodes the original spatial position of themicrosample within the biological specimen, thereby producing spatialencoded nucleic acids; sequencing the spatial encoded nucleic acids; andbased on the spatial information attached to each spatial encodednucleic acids, determining the original spatial location of the nucleicacid in the biological specimen.
 41. The method of claim 40, comprisingsequencing spatial encoding nucleic acids combined from a plurality ofdifferent microsamples in a single high throughput sequencing run.
 42. Asystem having a graphical user interface that presents, based on spatialinformation obtained from microsamples of a biological specimen, agraphical representation of the biological specimen including originalspatial position of a plurality of polynucleotides or polypeptides inthe biological specimen.
 43. A spatial preparation system configured toentrain in a fluidic stream a plurality of microsamples from abiological specimen, wherein the microsamples are contained in spatiallyseparated microdrops in a fluidic stream and positioned in an orderbased on their original spatial position within the biological specimen,wherein the system comprises: a) a spatial sampler subsystem configuredto extract a plurality of microsamples from different original spatialpositions in a biological specimen; and b) a spatial encoder subsystemcomprising one or more spatial encoder microchannels, each having aninlet and an outlet; wherein the spatial sampler subsystem delivers themicrosamples to the spatial encoder microchannel inlets in apredetermined order based on their original spatial position in thebiological specimen, and the spatial encoder subsystem incorporates themicrosamples into spatially separated microdrops in a fluidic stream.44. The spatial preparation system of claim 43, wherein: (i) the spatialsampler subsystem comprises: (1) a specimen holder, and (2) amultifunctional head comprising a transfer head comprising one or moreextraction channels, wherein the extraction channels communicate with aliquid source and, optionally, a gas source, each under positive and/ornegative pressure, and wherein the one or more extraction channelscomprise ends covered with one or more air permeable, cell impermeabletransfer membranes, and wherein, the multifunctional head is mounted ona three axis stage to position the multifunctional head to extract, bycontact adhesion or by vacuum, the microsamples from the specimen holderonto the one or more transfer membranes; and (ii) the spatial encodersubsystem comprises: (1) a microdroplet generator comprising a source ofimmiscible liquid in communication with each spatial encodermicrochannel at a junction, wherein mixture of the immiscible liquidwith the fluidic stream at the junction forms spatially separatedmicrodrops comprising the microsamples; and (2) optionally, amicrosample encoder assembly comprising a plurality of reservoirs, eachcomprising a different spatial marker and each communicating with thespatial encoder microchannel, and, optionally reservoirs comprising areactants sufficient to attach the tags to analytes in the microsamples,wherein different spatial markers are incorporated with microsamples indifferent microdrops.
 45. The system of claim 44, wherein themultifunctional head further comprises a dispense head configured todispense liquids, e.g., imaging reagents or dissociation solution, ontothe biological specimen.
 46. The system of claim 44, wherein thetransfer head comprises a plurality of extraction channels where in theextraction channels are arrayed in a two dimensional array (e.g., aline) or a three-dimensional array (e.g., a plane).
 47. The system ofclaim 46, wherein the spatial encoder subsystem comprises a plurality offluidic channels that merge into the encoder channel in which each hasan inlet configured to receive the microsamples from an extractionchannels.
 48. The system of claim 47, wherein, the transfer membraneshave attached thereto a plurality of capture elements, each captureelement comprising a particle, which is optionally paramagnetic, havingattached thereto one or more antibodies that bind into cells in thebiological specimen, and nucleic acid markers comprising positionalbarcodes comprising spatial information where the spatial informationcalling to the position of the particle on the multifunctional head. 49.The system of claim 48, wherein, the nucleic acid markers furthercomprise cell markers identifying the cell to which particle binds,and/or molecular barcodes that differently label different nucleic acidmolecules and a single cell.
 50. A spatial analysis system comprising:a) a spatial preparation subsystem of claim 44, and b) a spatiallibrarian subsystem configured to perform a series of biochemicalreactions on an emulsion comprising microdrops produced by the spatialpreparation subsystem, wherein the spatial librarian subsystemcomprises: a) a reaction device comprising an inlet configured toreceive microdrops from the spatial preparation subsystem, at least onereaction chamber, and an outlet; b) a reagent rail communicating withthe reaction device through a microchannel and comprising reagentsufficient to perform at least one of biochemical reaction on analytesin the microdrops; and c) one or more pumps configured to move thereagents from the reagent rail through the microchannel to the reactionchamber of the reaction device.
 51. The spatial analysis system of claim50, wherein the spatial librarian subsystem further comprising: c) atemperature controller configured to control temperature in the reactionchamber.
 52. The spatial analysis system of claim 50, wherein thespatial librarian subsystem further comprising: c) a magnet configuredto reversibly immobilize paramagnetic particles contained in thereaction chamber.
 53. The spatial analysis system of claim 50, whereinthe biochemical reactions comprise at least: (i) reverse transcriptionof messenger RNA into cDNA; and (ii) amplification of cDNA.
 54. Thespatial analysis system of claim 50, wherein the biochemical reactionscomprise at least: (i) primer extension of a primer hybridized to a DNAtemplate to create an extension product; and (ii) amplification of theextension product.
 55. A method comprising entraining in a fluidicstream a plurality of microsamples from a biological specimen, whereinthe microsamples are contained in spatially separated microdrops in thefluidic stream and positioned in an order based on their originalspatial position within the biological specimen.
 56. The method of claim55, comprising: a) providing a biological specimen; b) collectingmicrosamples from each of a plurality of different spatial positions inthe biological specimen; c) introducing the microsamples in apredetermined order into a fluidic stream in a fluidic channel; d)dividing the fluidic stream into microdrops by introducing boluses ofimmiscible liquid into the fluidic channel, whereby the microsamples areincorporated into microdrops that are spatially separated from eachother in the fluidic stream.
 57. The method of claim 55, furthercomprising: (i) introducing into the fluidic stream a plurality ofdifferent spatial markers encoding spatial information, wherein thedifferent spatial markers are incorporated into different microdrops inthe fluidic stream, thereby encoding each microdrop with spatialinformation.
 58. The method of claim 57, wherein the analytes comprisenucleic acids and the spatial markers comprise nucleic acids comprisingnucleic acid barcodes, wherein the method further comprises: (e)combining microdrops in a container in the form of an emulsion; (f)generating spatially tagged nucleic acids by tagging nucleic acidanalytes with the nucleic acid barcodes; (g) breaking the emulsion; (h)amplifying the tagged nucleic acids.
 59. The method of claim 58, whereinthe analytes comprise polyadenylated mRNA and the nucleic acid markersfurther comprise polyT tail, and generating spatially tagged nucleicacids comprises: hybridizing the polyT tail to polyadenylated mRNAnucleic acid markers to the mRNA molecules barcodes and reversetranscribing the polyadenylated messenger RNA to produce that spatiallytagged cDNA molecules; performing second strand synthesis on thespatially tagged cDNA molecules to produce tagged double stranded cDNAmolecules.
 60. The method of claim 58, wherein the analytes comprise DNAmolecules and the nucleic acid markers further comprise a nucleotidesequence complementary to a target sequence, and generating spatiallytagged nucleic acids comprises: hybridizing the complementary nucleotidesequence to a target sequence in the nucleic acid molecules andextending the nucleic acid markers to produce a double-stranded DNAmolecule.
 61. The method of claim 57, wherein further comprisingapplying imaging reagent to the biological sample; imaging thebiological sample to which the imaging reagent has been applied; basedon the imaging selecting features of interest at predetermined spatialpositions in the biological sample; and extracting the microsamplesincluding the selected features of interest.
 62. The method of claim 57,further comprising, based on spatial information encoded in themicrosamples determining the initial spatial position of the selectedfeatures in the biological specimen.